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Hai-Ying Chen,1,&ast; Ming-Chao Li,2,&ast; Dan Liao,1 Chi Li,1 Qiu-Ming Ji,1 Ping Guo,1 Zou Su,1 Yu-Hong Yang,1 Wen-Hao Xiao,1 Wen-Hui Zhai1 1Department of Psychiatry, Wuhan Wudong Hospital, Wuhan, People’s Republic of China; 2Department of Psychiatry, Wuhan Mental Health Center, Wuhan, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Dan Liao; Chi Li, Department of Psychiatry, Wuhan Wudong Hospital, No. 46 of Wudong Street, Qingshan District, Wuhan, 430084, People’s Republic of China, Tel +86 27 5052 8367, Fax +86 27 8643 8247, Email danliaodr@21cn.comObjective: Our study aimed to investigate the effects of computer-assisted cognitive remediation therapy (CCRT) on cognitive function, social function and quality of life in patients with vascular dementia (VD).Methods: Ninety-eight patients with VD were treated with CCRT in four 45-minute sessions per week over a course of 40 sessions to exercise four cognitive functions, including flexibility, working memory, plan execution and social cognition. The Mini-Mental State Examination (MMSE), Social Disability Screening Schedule (SDSS), Personal and Social Performance Scale (PSP), and Generic Quality of Life Inventory-74 (GQOL-74) were used to assess before and after treatment.Results: (1) The scores of orientation (5.60 ± 1.35), calculation (2.20 ± 0.79), verbal ability (7.10 ± 0.36), spatial ability (0.78 ± 0.42), immediate memory (2.42 ± 0.53), short-term memory (1.17 ± 0.78) and MMSE (23.36 ± 2.98) were all improved after treatment (P < 0.05) compared with those before treatment; (2) The scores of SDSS, PSP and Activities of Daily Living (ADL) after treatment were 8.23 ± 0.94, 81.36 ± 14.23, and 32.7 ± 12.1, and all of which improved (P < 0.05); (3) The scores of physical health were 68.24 ± 7.44, mental health were 69.75 ± 7.15, social function were 69.08 ± 7.43, material life were 37.46 ± 4.85 and the total score were 230.79 ± 9.56, all of which improved (P < 0.05).Conclusion: For patients with VD, CCRT can improve their cognitive function, social function, daily life ability and quality of life.Keywords: computer-assisted cognitive remediation, vascular dementia, cognitive function, social function, daily life ability

Yu-Tao Liu,1 Xue-Zhi Hao,1 De-Ruo Liu,2 Gang Cheng,3 Shu-Cai Zhang,4 Wen-Hua Xiao,5 Yi Hu,6 Jun-Feng Liu,7 Ming He,7 Cui-Min Ding,8 Li Zhang,9 Jun Wang,10 Hui Li,11 Gui-Lan Dong,12 Xiu-Yi Zhi,13 Jian Li,14 Yuan-Kai Shi1 1Department of Medical Oncology, Beijing Key Laboratory of Clinical Study on Anticancer Molecular Targeted Drugs, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, People’s Republic of China; 2Department of General Thoracic Surgery, China-Japan Friendship Hospital, Beijing, People’s Republic of China; 3Department of Medical Oncology, Beijing Hospital, Beijing, People’s Republic of China; 4Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, People’s Republic of China; 5Department of Oncology, The First Affiliated Hospital of Chinese PLA General Hospital, Beijing, People’s Republic of China; 6Department of Oncology, Chinese PLA General Hospital, Beijing, People’s Republic of China; 7Department of Thoracic Surgery, The Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei, People’s Republic of China; 8Department of Respiratory Medicine, The Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei, People’s Republic of China; 9Department of Respiratory Medicine, Peking Union Medical College Hospital, Affiliated to Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, People’s Republic of China; 10Department of Thoracic Surgery, Peking University People’s Hospital, Beijing, People’s Republic of China; 11Department of Thoracic Surgery, Beijing Chaoyang Hospital Affiliated to Capital Medical University, Beijing, People’s Republic of China; 12Department of Radiotherapy and Chemotherapy, Tangshan People’s Hospital, Tangshan, Hebei, People’s Republic of China; 13Department of Thoracic Surgery, Xuanwu Hospital of Capital Medical University, Beijing, People’s Republic of China; 14Department of Thoracic Surgery, Peking University First Hospital, Beijing, People’s Republic of ChinaCorrespondence: Yuan-Kai ShiDepartment of Medical Oncology, Beijing Key Laboratory of Clinical Study on Anticancer Molecular Targeted Drugs, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, People’s Republic of ChinaTel +86 10 8778 8293Email syuankai@cicams.ac.cnAbstract: The efficacy and possible role of epidermal growth factor receptor tyrosine kinase inhibitors in treating early-stage non-small-cell lung cancer have yet to be established. Therefore, we aimed to explore the efficacy and safety of icotinib in completely resected EGFR-mutant stage II–IIIA lung adenocarcinoma patients who underwent standard chemotherapy. This is a randomised, double-blinded, placebo-controlled, multicentre, Phase III trial. A total of 124 patients aged 18– 75 years who qualified the inclusion criteria were recruited. These patients were randomised (1:1) to receive either icotinib (125 mg 3 times per day) or placebo (the same dosage and frequency) for 36 months, followed by a further 36 months of observational window. The primary endpoint is disease-free survival (DFS), while the secondary endpoints are overall survival, 3-year and 5-year DFS, safety and tolerability of the medication, and health-related quality-of-life. Analyses will be conducted in a full analysis set and a per-protocol set as well. To our knowledge, the present study is the first randomised, double-blinded, placebo-controlled, multicenter trial designed to explore efficacy and safety of icotonib in this population. The results obtained in the near future may provide potential guidance in clinical practice.Trial Registration: This trial was registered on www.ClinicalTrail.gov as NCT02125240.Keywords: EGFR mutation, non-small-cell lung cancer, lung adenocarcinoma, icotinib, adjuvant chemotherapy

The thermal properties of interfaces in nanomaterials are critical for various technological applications, including thermal management in electronic and photonic devices, thermoelectric conversion and thermal insulation. Recent advancements in numerical simulation tools (the non-equilibrium Green's approach, the Boltzmann transport equation and the Monte Carlo method, molecular dynamics simulations) have significantly enhanced our understanding of phonon transport and scattering processes in nanomaterials. These advances have led to the discovery of new thermal interfacial materials and enabled precise modulation of phonon thermal conductance to achieve desired thermal performance. This review summarizes recent research progress in interface thermal transport, focusing on intriguing heat phenomena such as finite size effect and phonon coherent property. Additionally, it discusses strategies for modulating thermal conductance through disorder and roughness. Finally, the review proposes the opportunities and challenges associated with modulating interface thermal transport., (© 2024 IOP Publishing Ltd. All rights, including for text and data mining, AI training, and similar technologies, are reserved.)

The regulation of adipose tissue homeostasis is essential for maintaining energy and metabolism balance in the body. The peripheral nervous system plays a crucial role in this process. Previous related research primarily focused on the sympathetic nervous system and its release of norepinephrine, while recent attention has shifted to the field of adipose sensory nerves. Studies demonstrate that external stimuli can activate adipose sensory nerves through pathways involving transient receptor potential vanilloid-1 (TRPV1), adipokines, and fatty acids, thereby transmitting signals to the brain. Emerging techniques, such as adipose nerve imaging and denervation of tissues, have revealed the critical role of sensory nerves in the glucose and lipid metabolism, thermogenic function, and vascular regulation of adipose tissue. This article comprehensively reviews the latest research on the regulation and function of sensory nerves in adipose tissue, with a focus on the impact of metabolic diseases on adipose sensory nerves. This review discusses current issues and prospects on the mechanisms behind neural regulation in adipose tissue, hoping to contribute to a comprehensive understanding and providing directions for future research.

Testosterone is closely associated with lipid metabolism and known to affect body fat composition and muscle mass in males. However, the mechanisms by which testosterone acts on lipid metabolism are not yet fully understood, especially in teleosts. In this study, cyp17a1 -/- zebrafish ( Danio rerio ) exhibited excessive visceral adipose tissue (VAT), lipid content, and up-regulated expression and activity of hepatic de novo lipogenesis (DNL) enzymes. The assay for transposase accessible chromatin with sequencing (ATAC-seq) results demonstrated that chromatin accessibility of DNL genes was increased in cyp17a1 -/- fish compared to cyp17a1 +/+ male fish, including stearoyl-CoA desaturase ( scd ) and fatty acid synthase ( fasn ). Androgen response element (ARE) motifs in the androgen signaling pathway were significantly enriched in cyp17a1 +/+ male fish but not in cyp17a1 -/- fish. Both androgen receptor ( ar )-/- and wild-type (WT) zebrafish administered with Ar antagonist flutamide displayed excessive visceral adipose tissue, lipid content, and up-regulated expression and activity of hepatic de novo lipogenesis enzymes. The Ar agonist BMS-564929 reduced the content of VAT and lipid content, and down-regulated acetyl-CoA carboxylase a ( acaca ), fasn , and scd expression. Mechanistically, the rescue effect of testosterone on cyp17a1 -/- fish in terms of phenotypes was abolished when ar was additionally depleted. Collectively, these findings reveal that testosterone inhibits lipid deposition by down-regulating DNL genes via Ar in zebrafish, thus expanding our understanding of the relationship between testosterone and lipid metabolism in teleosts.

Objective: To evaluate the clinical utility value of questionnaires of Berlin, STOP, STOP-Bang (SBQ), Epworth Sleepiness Scale (ESS) in screening obstructive sleep apnea syndrome (OSAS) in pregnant women of different trimesters. Methods: Consecutive pregnant women at high risk for OSAS were enrolled from January, 2021 to April, 2022 at the obstetric clinic of Peking University People's Hospital. They completed questionnaires of Berlin, STOP, SBQ, ESS and also underwent an overnight polysomnography (PSG). To evaluate the accuracy of questionnaires of Berlin, STOP, SBQ, ESS, sensitivity, specificity, positive predictive values, negative predictive values and the area under the receiver operating characteristics (ROC) curve of these questionnaires in pregnancy across trimesters (Pregnancy 1-15 weeks was the first stage, pregnancy 16-27 weeks was the second stage, and pregnancy 28-40 weeks was the third stage) were calculated. Results: A total of 100 pregnant women [(34.5±4.3) years old (26-46 years old)] were included in this study, including 20, 35 and 45 pregnant women in the first, second and third trimester of pregnancy, respectively. Based on PSG results, 45 (45%) of 100 pregnant women were diagnosed with OSAS. The overall predictive values of the four questionnaires were not good, area under[AUC(95% CI )] the ROC curve ESS, Berlin questionnaire STOP and SBQ were 0.54(0.43, 0.66), 0.59 (0.47, 0.70), 0.62(0.51, 0.73) and 0.61 (0.49, 0.72), respectively, sensitivity was 35.6%, 65.9%, 48.9%, 28.9%, specificity was 71.7%, 52.5%, 73.6%, 92.5%. When categorized according to trimesters, the predicted values of the four questionnaires increased in the first trimester, the AUC (95% CI ) of STOP questionnaire was 0.81 (0.61, 1.00), sensitivity was 75.0%, specificity was 87.5%. Conclusion: The overall predictive power of the four screening questionnaires is limited in pregnant women. But predictive value of STOP questionnaire is acceptable in the first trimester.

Background: There are systematic differences in clinical features between women and men with schizophrenia (SCZ). The regulation of sex hormones may play a potential role in abnormal neurodevelopment in SCZ. Brain-derived neurotrophic factor (BDNF) and sex hormones have complex interacting actions that contribute to the etiology of SCZ., Aim: To investigate the influence of BDNF and sex hormones on cognition and clinical symptomatology in chronic antipsychotic-treated male SCZ patients., Methods: The serum levels of follicle-stimulating hormone, luteinizing hormone (LH), estradiol (E2), progesterone, testosterone (T), prolactin (PRL) and BDNF were compared between chronic antipsychotic-treated male (CATM) patients with SCZ ( n = 120) and healthy controls ( n = 120). The Positive and Negative Syndrome Scale was used to quantify SCZ symptoms, while neuropsychological tests were used to assess cognition. Neuropsychological tests, such as the Digit Cancellation Test (DCT), Semantic Verbal Fluency (SVF), Spatial Span Test (SS), Paced Auditory Serial Addition Test (PASAT), Trail Making Task (TMT-A), and Block Design Test (BDT), were used to assess executive functions (BDT), attention (DCT, TMT-A), memory (SS, PASAT), and verbal proficiency (SVF)., Results: Although E2 levels were significantly lower in the patient group compared to the healthy controls, T, PRL, and LH levels were all significantly higher. Additionally, the analysis revealed that across the entire sample, there were positive correlations between E2 Levels and BDNF levels as well as BDNF levels and the digital cancellation time. In CATM patients with SCZ, a significant correlation between the negative symptoms score and PRL levels was observed., Conclusion: Sex hormones and BDNF levels may also be linked to cognitive function in patients with chronic SCZ., Competing Interests: Conflict-of-interest statement: The authors declare no conflicts of interest., (©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved.)

β-hydroxybutyrate (β-HB), the most abundant ketone body, is produced primarily in the liver and acts as a substitute energy fuel to provide energy to extrahepatic tissues in the event of hypoglycemia or glycogen depletion. We now have an improved understanding of β-HB as a signal molecule and epigenetic regulatory factor as a result of intensive research over the last ten years. Because β-HB regulates various physiological and pathological processes, it may have a potential role in the treatment of metabolic diseases. The liver is the most significant metabolic organ, and the part that β-HB plays in liver disorders is receiving increasing attention. In this review, we summarize the therapeutic effects of β-HB on liver diseases and its underlying mechanisms of action. Moreover, we explore the prospects of exogenous supplements and endogenous ketosis including fasting, caloric restriction (CR), ketogenic diet (KD), and exercise as adjuvant nutritional therapies to protect the liver from damage and provide insights and strategies for exploring the treatment of various liver diseases., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Branched chain amino acids, as essential amino acids, can be used to synthesize nitrogen-containing compounds and also act as signal molecules to regulate substance metabolism. Studies have shown that the elevated level of branched chain amino acids is closely related to insulin resistance and type 2 diabetes. It can affect insulin signal transduction by activating mammalian target of rapamycin (mTOR) signal pathway, and regulate insulin resistance by damaging lipid metabolism and affecting mitochondrial function. In addition, abnormal catabolism of branched amino acids can lead to the accumulation of metabolic intermediates, such as branched chain α-keto acids, 3-hydroxyisobutyrate and β-aminoisobutyric acid. Branched chain α-keto acids and 3-hydroxyisobutyrate can induce insulin resistance by affecting insulin signaling pathway and damaging lipid metabolism. β-aminoisobutyric acid can improve insulin resistance by reducing lipid accumulation and inflammatory reaction and enhancing fatty acid oxidation. This paper systematically reviewed the regulatory effects and mechanisms of branched chain amino acids and their metabolic intermediates on insulin resistance, which will provide a new direction for the prevention and treatment of insulin resistance and type 2 diabetes.

General anesthesia shares many similarities with natural sleep in behavior and electroencephalogram (EEG) patterns. The latest evidence suggests that general anesthesia and sleep-wake behavior may share overlapping neural substrates. The GABAergic neurons in the basal forebrain (BF) have recently been demonstrated to play a key role in controlling wakefulness. It was hypothesized that BF GABAergic neurons may participate in the regulation of general anesthesia. Here, using in vivo fiber photometry, we found that the activity of BF GABAergic neurons was generally inhibited during isoflurane anesthesia, having obviously decreased during the induction of anesthesia and being gradually restored during the emergence from anesthesia, in Vgat-Cre mice of both sexes. Activation of BF GABAergic neurons with chemogenetic and optogenetic approaches decreased sensitivity to isoflurane, delayed induction, and accelerated emergence from isoflurane anesthesia. Optogenetic activation of BF GABAergic neurons decreased EEG δ power and the burst suppression ratio (BSR) during 0.8% and 1.4% isoflurane anesthesia, respectively. Similar to the effects of activating BF GABAergic cell bodies, photostimulation of BF GABAergic terminals in the thalamic reticular nucleus (TRN) also strongly promoted cortical activation and behavioral emergence from isoflurane anesthesia. Collectively, these results showed that the GABAergic BF is a key neural substrate for general anesthesia regulation that facilitates behavioral and cortical emergence from general anesthesia via the GABAergic BF-TRN pathway. Our findings may provide a new target for attenuating the depth of anesthesia and accelerating emergence from general anesthesia. SIGNIFICANCE STATEMENT The basal forebrain (BF) is a key brain region controlling sleep-wake behavior. Activation of GABAergic neurons in the BF potently promotes behavioral arousal and cortical activity. Recently, many sleep-wake-related brain structures have been reported to participate in the regulation of general anesthesia. However, it is still unclear what role BF GABAergic neurons play in general anesthesia. In this study, we aim to reveal the role of BF GABAergic neurons in behavioral and cortical emergence from isoflurane anesthesia and elucidate the underlying neural pathways. Understanding the specific role of BF GABAergic neurons in isoflurane anesthesia would improve our understanding of the mechanisms of general anesthesia and may provide a new strategy for accelerating emergence from general anesthesia., (Copyright © 2023 Cai, Su et al.)

Lower limb osteoarthritis (OA) is a chronic, multifactorial disease characterized by impaired physical function, chronic pain, compromised psychological health and decreased social functioning. Chronic inflammation plays a critical role in the pathophysiology of OA. Tai Chi is a type of classical mind-body exercise derived from ancient Chinese martial arts. Evidence supports that Tai Chi has significant benefits for relieving lower limb OA symptoms. Using a biopsychosocial framework, this review aims to elucidate the beneficial effects of Tai Chi in lower limb OA and disentangle its potential mechanisms from the perspective of biology, psychology, and social factors. Complex biomechanical, biochemical, neurological, psychological, and social mechanisms, including strengthening of muscles, proprioception improvement, joint mechanical stress reduction, change of brain activation and sensitization, attenuation of inflammation, emotion modulation and social support, are discussed., (© 2021. The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag GmbH Germany, part of Springer Nature.)

Aim: To elucidate the mechanism of miR-128-3p in peritoneal fibrosis (PF)., Methods: Peritoneal mesothelial cells (PMCs) were dealt with high glucose (HG) for 3 days. The expressions of miR-128-3p, p21-activated kinase 2 (PAK2), spleen tyrosine kinase (SyK), and transforming growth factor-β1 (TGF-β1) were detected with quantitative real-time reverse transcription polymerase chain reaction. The levels of IL-1β, TNF-α, IL-6, and monocyte chemotactic protein-1 in supernatant were measured by ELISA. Proteins of TGF-β1, SyK, PAK2, α-SMA, collagen I, vimentin, ERK/AP-1, and IκBα/NF-κB pathway related proteins were measured by Western blot. The correlation between miR-128-3p and PAK2 was found by bioinformatics analysis and luciferase reporter gene analysis., Results: miR-128-3p was decreased while PAK2, SyK, and TGF-β1 were increased in HG-induced PMCs. Moreover, miR-128-3p inhibited HG-induced fibrosis and inflammation in PMCs by targeting PAK2. PAK2 activated SyK, which induced TGF-β1 expression through ERK/AP-1 and IκBα/NF-κB pathways to promote HG-induced fibrosis of PMCs., Conclusion: miR-128-3p inhibited HG-induced PMCs fibrosis via PAK2/SyK/TGF-β1 axis., (© 2022 International Society for Apheresis and Japanese Society for Apheresis.)

The lateral hypothalamus (LH) is an important brain region mediating sleep-wake behavior. Recent evidence has shown that astrocytes in central nervous system modulate the activity of adjacent neurons and participate in several physiological functions. However, the role of LH astrocytes in sleep-wake regulation remains unclear. Here, using synchronous recording of electroencephalogram/electromyogram in mice and calcium signals in LH astrocytes, we show that the activity of LH astrocytes is significantly increased during non-rapid eye movement (NREM) sleep-to-wake transitions and decreased during Wake-to-NREM sleep transitions. Chemogenetic activation of LH astrocytes potently promotes wakefulness and maintains long-term arousal, while chemogenetic inhibition of LH astrocytes decreases the total amount of wakefulness in mice. Moreover, by combining chemogenetics with fiber photometry, we show that activation of LH astrocytes significantly increases the calcium signals of adjacent neurons, especially among GABAergic neurons. Taken together, our results clearly illustrate that LH astrocytes are a key neural substrate regulating wakefulness and encode this behavior through surrounding GABAergic neurons. Our findings raise the possibility that overactivity of LH astrocytes may be an underlying mechanism of clinical sleep disorders., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The dorsal raphe nucleus (DRN) has previously been proved to be involved in the regulation of the sleep-wake behavior. DRN contains several neuron types, such as 5-HTergic and GABAergic neurons. GABAergic neurons, which are the second largest cell subtype in the DRN, participate in a variety of neurophysiological functions. However, their role in sleep-wake regulation and the underlying neural circuitry remains unclear. Herein, we used fiber photometry and synchronous electroencephalogram (EEG)/electromyography (EMG) recording to demonstrate that DRN GABAergic neurons exhibit high activities during wakefulness and low activities during NREM sleep. Short-term optogenetic activation of DRN GABAergic neurons reduced the latency of NREM-to-wake transition and increased the probability of wakefulness, while long-term optogenetic activation of these neurons significantly increased the amount of wakefulness. Chemogenetic activation of DRN GABAergic neurons increased wakefulness for almost 2 h and maintained long-lasting arousal. In addition, inhibition of DRN GABAergic neurons with chemogenetics caused a reduction in the amount of wakefulness. Finally, similar to the effects of activating the soma of DRN GABAergic neurons, optogenetic stimulation of their terminals in the ventral tegmental area (VTA) induced instant arousal and promoted wakefulness. Taken together, our results illustrated that DRN GABAergic neurons are vital to the induction and maintenance of wakefulness, which promote wakefulness through the GABAergic DRN-VTA pathway., (© The Author(s) 2022. Published by Oxford University Press on behalf of Sleep Research Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: To evaluate the performance of a microfluidic platelet function test platform (MPFTP) previously established by our research group., Methods: The effects of flow shear rate and storage time on platelet function test were analyzed taking the MPFTP as the object. The intra-assay variability of the MPFTP was evaluated. The function of platelet in peripheral venous blood from 24 healthy volunteers was assessed using the MPFTP and light transmission turbidity, either in the presence of 20 μmol/L acetylsalicylic acid (AS, an inhibitor of cyclooxygenase 1) or 50 μmol/L 5-phospho-2-methylthioadenosine (2-MeSAMP, a P2Y 12 receptor inhibitor). The diagnostic performance of both methods in assaying platelet function inhibition by AS and 2-MeSAMP was analyzed by using receiver operating characteristic (ROC) curve., Results: Under the flow shear rate of 1 500 s -1 , our MPFTP could dynamically monitor platelet adhesion and aggregation, as well as quantify platelet function. Platelet aggregation increased with the increase of flow shear rate, while sample storage at room temperature for up to 5 h did not affect results of platelet function test. The intra-assay variability coefficient of variation of the MPFTP was <15%. The area under the curve of ROC showed that this platform had good diagnostic performance in the identification of platelet function inhibition by AS and 2-MeSAMP., Conclusion: This MPFTP shows good analytical performance for the assay of platelet function and can be developed into a new clinical platelet function test device in the future.

Skeletal muscle is the largest organ of human body, which completes 80%-90% of glucose intake stimulated by insulin, and is closely related to the occurrence and development of insulin resistance (IR). Skeletal muscle is one of the main places of lipid metabolism, and lipid metabolites participate in skeletal muscle metabolism as signal molecules. Fatty acids regulate skeletal muscle insulin sensitivity through insulin signaling pathway, inflammatory response and mitochondrial function. Saturated fatty acids (SFAs) induce insulin resistance by impairing insulin signal transduction, inducing mitochondrial dysfunction and inflammatory response, while unsaturated fatty acids reverse the adverse effects of SFAs and ameliorate IR by enhancing insulin signal transduction and anti-inflammatory effect. In addition, disorders of lipid metabolism in skeletal muscle cause accumulation of harmful metabolic intermediates, such as diacylglycerol, ceramide and long-chain acyl-coenzyme A, and induce IR by directly or indirectly damaging insulin signaling pathway. This article reviews the research progress of lipid metabolic intermediates regulating insulin sensitivity in skeletal muscle, which will help to better understand the pathogenesis of diabetes.

Crocetin, a high-value apocarotenoid in saffron, is widely applied to the fields of food and medicine. However, the existing method of obtaining crocetin through large-scale cultivation is far from meeting the market demand. Microbial synthesis of crocetin is a potential alternative to traditional resources, and it is found that carotenoid cleavage dioxygenase (CCD) is the critical enzyme to synthesize crocetin. So, in this study, we used "hybrid-tunnel" engineering to obtain variants of Crocus sativus -derived Cs CCD2, essential for zeaxanthin conversion into crocetin, with a broader substrate specificity and higher catalytic efficiency. Variants including S323A, with a lower charge bias and a larger tunnel size than the wild-type, showed a 5-fold higher crocetin titer in yeast-based fermentations. S323A could also convert the β-carotene substrate to crocetin dialdehyde and exhibited a 12.83-fold greater catalytic efficiency ( k cat / K m ) toward zeaxanthin than the wild-type in vitro . This strategy enabled the production of 107 mg/L crocetin in 5 L fed-batch fermentation, higher than that previously reported. Our findings demonstrate that engineering access tunnels to expand the substrate profile by in silico protein design represents a viable strategy to refine the catalytic properties of enzymes across a range of applications.

7-Dehydrocholesterol (7-DHC) is the direct precursor to manufacture vitamin D 3 . Our previous study has achieved 7-DHC synthesis in Saccharomyces cerevisiae based on the endogenous post-squalene pathway. However, the distribution of post-squalene enzymes between the endoplasmic reticulum (ER) and lipid bodies (LD) might raise difficulties for ERG proteins to catalyze and deliver sterol intermediates, resulting in unbalanced metabolic flow and low product yield. Herein, we intended to rearrange the subcellular location of post-squalene enzymes to alleviate metabolic bottleneck and boost 7-DHC production. After identifying the location of DHCR24 (C-24 reductase, the only heterologous protein for 7-DHC biosynthesis) on ER, all the ER-located enzymes were grouped into four modules: ERG1/11/24, ERG25/26/27, ERG2/3, and DHCR24. These modules attempted to be overexpressed either on ER or on LDs. As a result, expression of LD-targeted DHCR24 and ER-located ERG1/11/24 could promote the conversion efficiency among the sterol intermediates to 7-DHC, while locating module ERG2/3 into LDs improved the whole metabolic flux of the post-squalene pathway. Coexpressing LD-targeted ERG2/3 and DHCR24 (generating strain SyBE&#95;Sc01250035) improved 7-DHC production from 187.7 to 308.2 mg/L at shake-flask level. Further expressing ER-targeted module ERG1/11/24 in strain SyBE&#95;Sc01250035 dramatically reduced squalene accumulation from 620.2 mg/L to the lowest level (by 93.8%) as well as improved 7-DHC production to the highest level (to 342.2 mg/L). Then targeting module ERG25/26/27 to LDs further increased 7-DHC titer to 360.6 mg/L, which is the highest shake-flask level production for 7-DHC ever reported. Our study not only proposes and further proves the concept of pathway compartmentalized reconstitution to regulate metabolic flux but also provides a promising chassis to produce other steroidal compounds through the post-squalene pathway., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Guo, Yao, Xiao, Wang, Zhao and Yuan.)

In Saccharomyces cerevisiae , conventional 2μ-plasmid based plasmid (pC2μ, such as pRS425) have been widely adopted in pathway engineering for multi-copy overexpression of key genes. However, the loss of partition and copy number control elements of yeast endogenous 2μ plasmid (pE2μ) brings the issues concerning plasmid stability and copy number of pC2μ, especially in long-term fermentation. In this study, we developed a method based on CRISPR/Cas9 to edit pE2μ and built the pE2μ multi-copy system by insertion of the target DNA element and elimination of the original pE2μ plasmid. The resulting plasmid pE2μRAF1 and pE2μREP2 demonstrated higher copy number and slower loss rate than a pC2μ control plasmid pRS425RK, when carrying the same target gene. Then, moving the essential gene TPI1 (encoding triose phosphate isomerase) from chromosome to pE2μRAF1 could increase the plasmid viability to nearly 100% and further increase the plasmid copy number by 73.95%. The expression using pE2μ multi-copy system demonstrated much smaller cell-to-cell variation comparing with pC2μ multi-copy system. With auxotrophic complementation of TPI1 , the resulting plasmid pE2μRT could undergo cultivation of 90 generations under non-selective conditions without loss. Applying pE2μ multi-copy system for dihydroartemisinic acid (DHAA) biosynthesis, the production of DHAA was increased to 620.9 mg/L at shake-flask level in non-selective rich medium. This titer was 4.73-fold of the strain constructed based on pC2μ due to the more stable pE2μ plasmid system and with higher plasmid copy number. This study provides an improved expression system in yeast, and set a promising platform to construct biosynthesis pathway for valuable products., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zeng, Yao, Xiao, Luo, Wang and Yuan.)

Aberrant activation of the cholesterol biosynthesis supports tumor cell growth. In recent years, significant progress has been made by targeting rate-limiting enzymes in cholesterol biosynthesis pathways to prevent carcinogenesis. However, precise mechanisms behind cholesterol degradation in cancer cells have not been comprehensively investigated. Here, we report that codon optimization of the orthologous cholesterol 7-desaturase, NVD-BM from Bombyx mori , significantly slowed melanoma cell proliferation and migration, and inhibited cancer cell engraftment in nude mice, by converting cholesterol to toxic 7-dehydrocholesterol. Based on these observations, we established a synthetic genetic circuit to induce melanoma cell regression by sensing tumor specific signals in melanoma cells. The dual-input signals, RELA proto-oncogene (RELA) and signal transducer and activator of transcription 1 (STAT1), activated NVD-BM expression and repressed melanoma cell proliferation and migration. Mechanically, we observed that NVD-BM decreased Akt1-ser473 phosphorylation and inhibited cytoplasmic RELA translocation. Taken together, NVD-BM was identified as a tumor suppressor in malignant melanoma, and we established a dual-input biosensor to promote cancer cell regression, via Akt1/NF-κB signaling. Our results demonstrate the potential therapeutic effects of cholesterol 7-desaturase in melanoma metabolism, and provides insights for genetic circuits targeting 7-dehydrocholesterol accumulation in tumors.

Carotenoids, a variety of natural products, have significant pharmaceutical and commercial potential. Phytoene dehydrogenase (CrtI) is the rate-limit enzyme for carotenoid synthesis, whose catalysis specificity results in various carotenoids. However, the structural characteristics of CrtI for controlling the catalysis specificity on dehydrogenation steps are still unclear, which limited the development of CrtI function. Here we confirmed two mutation sites H136 and H453 in the mutant library of CrtI from Blakeslea trispora , which markedly regulated catalytic specificity. Interestingly, the sequence alignment features at H136 and H453 were consistent with the phylogenetic analysis of CrtI families. Subsequently, the functions of saturated mutants at H136 and H453 were clustered by principal component analysis (PCA) and k-means. According to the clustering results, diversiform mutants with specific dehydrogenation function provided important application value for carotenoid product customization. Meanwhile, this study also enriched the theory of enzyme evolution and guided the functional development of enzymes.

When attacked by herbivores, plants emit volatiles to attract parasitoids and predators of herbivores. However, our understanding of the effect of plant volatiles on the subsequent behaviour of conspecific parasitoids when herbivores on plants are parasitized is limited. In this study, rice plants were infested with gravid females of the brown planthopper (BPH) Nilaparvata lugens for 24 hr followed by another 24 hr in which the BPH eggs on plants were permitted to be parasitized by their egg parasitoid, Anagrus nilaparvatae; volatiles from rice plants that underwent such treatment were less attractive to subsequent conspecific parasitoids compared to the volatiles from plants infested with gravid BPH females alone. Chemical analysis revealed that levels of JA and JA-Ile as well as of four volatile compounds-linalool, MeSA, α-zingiberene and an unknown compound-from plants infested with BPH and parasitized by wasps were significantly higher than levels of these compounds from BPH-infested plants. Laboratory and field bioassays revealed that one of the four increased chemicals-α-zingiberene-reduced the plant's attractiveness to the parasitoid. These results suggest that host plants can fine-tune their volatiles to help egg parasitoids distinguish host habitats with parasitized hosts from those without., (© 2020 John Wiley & Sons Ltd.)

Direct bioproduction of DHAA (dihydroartemisinic acid) rather than AA (artemisinic acid), as suggested by previous work would decrease the cost of semi-biosynthesis artemisinin by eliminating the step of initial hydrogenation of AA. The major challenge in microbial production of DHAA is how to efficiently manipulate consecutive key enzymes ADH1 (artemisinic alcohol dehydrogenase), DBR2 [artemisinic aldehyde Δ11(13) reductase] and ALDH1 (aldehyde dehydrogenase) to redirect metabolic flux and elevate the ratio of DHAA to AA (artemisinic acid). Herein, DHAA biosynthesis was achieved in Saccharomyces cerevisiae by introducing a series of heterologous enzymes: ADS (amorpha-4,11-diene synthase), CYP71AV1 (amorphadiene oxidase), ADH1, DBR2 and ALDH1, obtaining initial DHAA/AA ratio at 2.53. The flux toward DHAA was enhanced by pairing fusion proteins DBR2-ADH1 and DBR2-ALDH1, leading to 1.75-fold increase in DHAA/AA ratio (to 6.97). Moreover, to promote the substrate preference of ALDH1 to dihydroartemisinic aldehyde (the intermediate for DHAA synthesis) over artemisinic aldehyde (the intermediate for AA synthesis), two rational engineering strategies, including downsizing the active pocket and enhancing the stability of enzyme/cofactor complex, were proposed to engineer ALDH1. It was found that the mutant H194R, which showed better stability of the enzyme/NAD + complex, obtained the highest DHAA to AA ratio at 3.73 among all the mutations. Then the mutant H194R was incorporated into above rebuilt fusion proteins, resulting in the highest ratio of DHAA to AA (10.05). Subsequently, the highest DHAA reported titer of 1.70 g/L (DHAA/AA ratio of 9.84) was achieved through 5 L bioreactor fermentation. The study highlights the synergy of metabolic engineering and protein engineering in metabolic flux redirection to get the most efficient product to the chemical process, and simplified downstream conversion process., (Copyright © 2020 Zeng, Yao, Wang, Xiao and Yuan.)

As age increases, the risk of developing type 2 diabetes increases, which is associated with senile skeletal muscle dysfunction. During skeletal muscle aging, mitochondrial dysfunction, intramyocellular lipid accumulation, increased inflammation, oxidative stress, modified activity of insulin sensitivity regulatory enzymes, endoplasmic reticulum stress, decreased autophagy, sarcopenia and over-activated renin-angiotensin system may occur. These changes can impair skeletal muscle insulin sensitivity and increase the risk of insulin resistance and type 2 diabetes during skeletal muscle aging. This review of the mechanism of the increased risk of insulin resistance during skeletal muscle aging will provide a more comprehensive explanation for the increased incidence of type 2 diabetes in elderly individuals, and will also provide a more comprehensive perspective for the prevention and treatment of type 2 diabetes in elderly populations., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

Many natural and man-made urban lakes have been developed under urbanization. A unique feature of these lakes is the lack of an outlet; thus, they are defined as urban closed lakes (UCLs). UCLs are facing unexpected eutrophication under climate change and human activities. Our study assessed the trophic state, assimilative capacity (AC) and pollutant reduction of UCLs under different precipitation frequencies in Wuhan, China based on Carlson's Trophic State Index, assimilative capacity modelling, field investigations and observed data. The UCLs in Wuhan are nearly eutrophic in summer. Three primary nutrient sources are atmospheric deposition, pollutants carried in rainfall and nutrients released by sediments. TN and TP in the UCL water column are primarily contributed by surface runoff. The ACs of TN and TP in 2015 for Lingjiao Lake, Yue Lake, and Houxianghe Lake were 3472.07 kg, 13,800.99 kg, and 2805.58 kg, respectively, and 641.66 kg, 8386.79 kg, and 800.14 kg, respectively. The ACs of TN and TP were much higher at a 25% precipitation frequency (wet year) compared with a 50% frequency, and the lowest AC was observed at a 75% precipitation frequency (dry year). A comparison of the pollution load and AC showed that TN and TP reduction was highest in the dry and wet years, respectively. We found that specific meteorological conditions in the early stage led to the algal bloom. These results can facilitate governmental decision making in the future., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The skeletal muscle mass accounts for more than 40% of the body weight of healthy adults. The skeletal muscle not only plays an important role in physical activities but also affects the function of other organs as a secretory organ secreting multiple muscle factors. Therefore, it is important to maintain the normal quantity and function of skeletal muscle. Skeletal muscle mass is the basis of skeletal muscle function and is often affected by many factors such as exercise and disease. Resistance exercise training induces increased protein synthesis in skeletal muscle cells, while limb disuse, chronic obstructive pulmonary disease, heart failure, chronic kidney disease, cachexia, Duchenne muscular dystrophy and many other pathological conditions lead to decreased protein synthesis or enhanced protein degradation of skeletal muscle cells. The process of skeletal muscle hypertrophy involves changes in multiple signaling pathways, such as IGF-1/PI3K/Akt, myostatin and G protein. On the other hand, activations of the ubiquitin-proteasome system, IGF-1/Akt/FoxO, autophagy-lysosomal pathway, NF-κB, and the glucocorticoid-mediated signaling pathways play important roles in regulating muscle atrophy. These signaling pathways regulate skeletal muscle mass and are modulated by some different conditions. This review briefly summarizes the signaling pathways of skeletal muscle mass control.

Insulin resistance is a common pathophysiological mechanism of obesity and type 2 diabetes mellitus. Skeletal muscle is one of the major target organs of insulin-mediated glucose uptake, metabolism and utilization, and it is the earliest and most important site of insulin resistance. Studies have shown that the impairments of glucose uptake, insulin signaling pathway and mitochondrial biosynthesis are closely related to skeletal muscle insulin resistance. When insulin resistance develops in skeletal muscle, multiple microRNAs (miRNAs) are up-regulated (miR-106b, miR-23a, miR-761, miR-135a, Let-7 and miR-29a) or down-regulated (miR-133a, miR-149 and miR-1). They participate in the regulation of skeletal muscle glucose uptake, insulin signaling pathway and mitochondrial biogenesis, and thus play important roles in the occurrence and development of skeletal muscle insulin resistance. Therefore, these miRNAs may serve as potential targets for the treatment of skeletal muscle insulin resistance or diabetes.

The toxic catabolic intermediates of branched chain amino acids can cause insulin resistance, and are involved in different mechanisms in different metabolic tissues. In skeletal muscle, 3-hydroxy-isobutyrate produced by valine promotes skeletal muscle fatty acid uptake, resulting in the accumulation of incompletely oxidized lipids in skeletal muscle, causing skeletal muscle insulin resistance. In the liver, branched-chain α-keto acids decompose in large amounts, promote hepatic gluconeogenesis, and lead to the accumulation of multiple acylcarnitines, which damages the mitochondrial tricarboxylic acid cycle, resulting in the accumulation of incomplete oxidation products, oxidative stress in mitochondria, and hepatic insulin resistance. In adipose tissue, the expression of branched-chain amino acid catabolic enzymes (branched-chain amino acid transaminase, branched-chain α-keto acid dehydrogenase) is reduced, resulting in an increased level of plasma branched-chain amino acids, thereby causing massive decomposition of branched-chain amino acids in tissues such as skeletal muscle and liver, and inducing insulin resistance. However, branched-chain amino acids, as a common nutritional supplement for athletes, do not induce insulin resistance. A possible explanation for this phenomenon is that exercise can enhance the mitochondrial oxidative potential of branched-chain amino acids, alleviate or even eliminate the accumulation of branched-chain amino acid catabolic intermediates, and promotes branched-chain amino acids catabolism into beta-aminoisobutyric acid, increasing plasma beta-aminoisobutyric acid concentration, improving insulin resistance. This article reveals the mechanism of BCAA-induced insulin resistance and the relationship between exercise and BCAAs metabolism, adds a guarantee for the use of BCAAs, and provides a new explanation for the occurrence of diabetes and how exercise improves diabetes.

The decline in skeletal muscle mass and function with age is referred as sarcopenia. It is characterized by the muscle fiber's quality, strength, muscle endurance and metabolic ability decreasing as well as the fat and connective tissue growing. Previous studies have shown that sarcopenia in itself features decreased number and cross-sectional area of muscle fibers and the net degradation of protein, which results from the joint effects of multiple factors such as the exacerbation of inflammation, oxidative stress injury, mitochondrial dysfunction, abnormal autophagy and dysregulation of muscle quality regulatory factors. In this review, we systematically displayed the molecular mechanism of sarcopenia, which will be helpful to deepen our understanding of sarcopenia and provide potential targets for the prevention and treatment of sarcopenia.

Background: 7-Dehydrocholesterol (7-DHC) has attracted increasing attentions due to its great medical value and the enlarging market demand of its ultraviolet-catalyzed product vitamin D 3 . Microbial production of 7-DHC from simple carbon has been recognized as an attractive complement to the traditional sources. Even though our previous work realized 7-DHC biosynthesis in Saccharomyces cerevisiae , the current productivity of 7-DHC is still too low to satisfy the demand of following industrialization. As increasing the compatibility between heterologous pathway and host cell is crucial to realize microbial overproduction of natural products with complex structure and relative long pathway, in this study, combined efforts in tuning the heterologous Δ 24 -dehydrocholesterol reductase (DHCR24) and manipulating host cell were applied to promote 7-DHC accumulation., Results: In order to decouple 7-DHC production with cell growth, inducible GAL promoters was employed to control 7-DHC synthesis. Meanwhile, the precursor pool was increased via overexpressing all the mevalonate (MVA) pathway genes ( ERG10 , ERG13 , tHMG1 , ERG12 , ERG8 , ERG19 , IDI1 , ERG20 ). Through screening DHCR24s from eleven tested sources, it was found that DHCR24 from Gallus gallus ( Gg &#95;DHCR24) achieved the highest 7-DHC production. Then 7-DHC accumulation was increased by 27.5% through stepwise fine-tuning the transcription level of Gg &#95;DHCR24 in terms of altering its induction strategy, integration position, and the used promoter. By blocking the competitive path (Δ ERG6 ) and supplementing another copy of Gg &#95;DHCR24 in locus ERG6 , 7-DHC accumulation was further enhanced by 1.07-fold. Afterward, 7-DHC production was improved by 48.3% (to 250.8 mg/L) by means of deleting NEM1 that was involved in lipids metabolism. Eventually, 7-DHC production reached to 1.07 g/L in 5-L bioreactor, which is the highest reported microbial titer as yet known., Conclusions: Combined engineering of the pathway and the host cell was adopted in this study to boost 7-DHC output in the yeast. 7-DHC titer was stepwise improved by 26.9-fold compared with the starting strain. This work not only opens large opportunities to realize downstream de novo synthesis of other steroids, but also highlights the importance of the combinatorial engineering of heterologous pathway and host to obtain microbial overproduction of many other natural products.

Escherichia coli KO11 is a popular ethanologenic strain, but is more sensitive to ethanol than other producers. Here, an ethanol-tolerant mutant EM was isolated from ultraviolet mutagenesis library of KO11. Comparative genomic analysis added by piecewise knockout strategy and complementation assay revealed EKO11&#95;3023 (espA) within the 36.6-kb deletion from KO11 was the only locus responsible for ethanol sensitivity. Interestingly, when espA was deleted in strain W (the parent strain of KO11), ethanol tolerance was dramatically elevated to the level of espA-free hosts [e.g., MG1655 and BL21(DE3)]. And overexpression of espA in strains MG1655 and BL21(DE3) led to significantly enhanced ethanol sensitivity. In addition to ethanol, deletion of espA also improved cell tolerance to other short-chain (C2-C4) alcohols, including methanol, isopropanol, n-butanol, isobutanol and 2-butanol. Therefore, espA was responsible for short-chain alcohol sensitivity of W-strains compared to other cells, which provides a potential engineering target for alcohols production.

Seed dormancy and germination are regulated by complex mechanisms controlled by diverse hormones and environmental cues. Abscisic acid (ABA) promotes seed dormancy and inhibits seed germination and post-germination growth. Calmodulin (CaM) signals are involved with the inhibition of ABA during seed germination and seedling growth. In this study, we showed that Arabidopsis thaliana IQM4 could bind with calmodulin 5 (CaM5) both in vitro and in vivo , and that the interaction was the Ca 2+ -independent type. The IQM4 protein was localized in the chloroplast and the IQM4 gene was expressed in most tissues, especially the embryo and germinated seedlings. The T-DNA insertion mutants of IQM4 exhibited the reduced primary seed dormancy and lower ABA levels compared with wild type seeds. Moreover, IQM4 plays key roles in modulating the responses to ABA, salt, and osmotic stress during seed germination and post-germination growth. T-DNA insertion mutants exhibited ABA-insensitive and salt-hypersensitive phenotypes during seed germination and post-germination growth, whereas IQM4 -overexpressing lines had ABA- and osmotic-hypersensitive, and salt-insensitive phenotypes. Gene expression analyses showed that mutation of IQM4 inhibited the expression of ABA biosynthetic genes NCED6 and NCED9 , and seed maturation regulators LEC1, LEC2, ABI3 , and ABI5 during the silique development, as well as promoted the expression of WRKY40 and inhibited that of ABI5 in ABA-regulated seed germination. These observations suggest that IQM4 is a novel Ca 2+ -independent CaM-binding protein, which is positively involved with seed dormancy and germination in Arabidopsis .

The power of synthetic biology has enabled the expression of heterologous pathways in cells, as well as genome-scale synthesis projects. The complexity of biological networks makes rational de novo design a grand challenge. Introducing features that confer genetic flexibility is a powerful strategy for downstream engineering. Here we develop an in vitro method of DNA library construction based on structural variation to accomplish this goal. The "in vitro SCRaMbLE system" uses Cre recombinase mixed in a test tube with purified DNA encoding multiple loxPsym sites. Using a β-carotene pathway designed for expression in yeast as an example, we demonstrate top-down and bottom-up in vitro SCRaMbLE, enabling optimization of biosynthetic pathway flux via the rearrangement of relevant transcription units. We show that our system provides a straightforward way to correlate phenotype and genotype and is potentially amenable to biochemical optimization in ways that the in vivo system cannot achieve.

SCRaMbLE (Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution) is a genome restructuring technique that can be used in synthetic genomes such as that of Sc2.0, the synthetic yeast genome, which contains hundreds to thousands of strategically positioned loxPsym sites. SCRaMbLE has been used to induce rearrangements in yeast strains harboring one or more synthetic chromosomes, as well as plasmid DNA in vitro and in vivo. Here we describe a collection of heterozygous diploid strains produced by mating haploid semisynthetic Sc2.0 strains to haploid native parental strains. We subsequently demonstrate that such heterozygous diploid strains are more robust to the effects of SCRaMbLE than haploid semisynthetic strains, rapidly improve rationally selected phenotypes in SCRaMbLEd heterozygous diploids, and establish that multiple sets of independent genomic rearrangements are able to lead to similar phenotype enhancements. Finally, we show that heterozygous diploid SCRaMbLE can also be carried out in interspecies hybrid strains.

The development of chemotherapy-induced painful peripheral neuropathy is a major dose-limiting side effect of many chemotherapeutics, including bortezomib, but the mechanisms remain poorly understood. We now report that bortezomib causes the dysregulation of de novo sphingolipid metabolism in the spinal cord dorsal horn to increase the levels of sphingosine-1-phosphate (S1P) receptor 1 (S1PR1) ligands, S1P and dihydro-S1P. Accordingly, genetic and pharmacological disruption of S1PR1 with multiple S1PR1 antagonists, including FTY720, blocked and reversed neuropathic pain. Mice with astrocyte-specific alterations of S1pr1 did not develop neuropathic pain and lost their ability to respond to S1PR1 inhibition, strongly implicating astrocytes as a primary cellular substrate for S1PR1 activity. At the molecular level, S1PR1 engaged astrocyte-driven neuroinflammation and altered glutamatergic homeostasis, processes blocked by S1PR1 antagonism. Our findings establish S1PR1 as a target for therapeutic intervention and provide insight into cellular and molecular pathways. As FTY720 also shows promising anticancer potential and is FDA approved, rapid clinical translation of our findings is anticipated., (© 2018 Stockstill et al.)

The objective of this study was to explore the roles of macrophages in the regeneration of injured skeletal muscle and the mechanisms involved. Mice were randomly divided into the following groups: muscle contusion (S), muscle contusion control (S Con ), macrophages depleted (T) and macrophages depleted control (T Con ) groups. Muscle contusion model was created by high-energy blunt injury. Macrophages depletion model was constructed by injection of clodronate-liposomes. Their gastrocnemius muscles were harvested at the time points of 1, 3, 7 and 14 d post-injury. The changes in skeletal muscle morphology were assessed by hematoxylin-eosin (HE) staining and Masson's trichrome staining. The mRNA and protein levels of inflammatory cytokines, chemokines and oxidative stress factors were analyzed by real-time polymerase chain reaction (RCR) and Western blotting, respectively. HE staining results showed that a small amount of regenerating myofibers were observed in the S group (14 d post-injury), whereas a large number of regenerating muscle fibers were observed in the T group. Quantitative analyses showed that the sizes of regenerating myofibers were significantly smaller in the T group as compared with the S group at 14 d post-injury (P < 0.05). At the same time, Masson staining results showed that macrophage depletion significantly increased the area of fibrosis as compared with the S group at 14 d post-injury (P < 0.01). The expression levels of inflammatory cytokines, chemokines, and oxidative stress factors were increased significantly after muscle injury. Moreover, macrophage depletion increased the expressions of inflammatory cytokines, chemokines and oxidative stress factors as compared with the S group during the later stage of injury (7-14 d post-injury). These results suggest that macrophages depletion can aggravate fibrosis and impair muscle regeneration, and inflammatory cytokines, chemokines and oxidative stress factors may be involved in this process.

Rapid and highly efficient mating-type switching of Saccharomyces cerevisiae enables a wide variety of genetic manipulations, such as the construction of strains, for instance, isogenic haploid pairs of both mating-types, diploids and polyploids. We used the CRISPR/Cas9 system to generate a double-strand break at the MAT locus and, in a single cotransformation, both haploid and diploid cells were switched to the specified mating-type at ∼80% efficiency. The mating-type of strains carrying either rod or ring chromosome III were switched, including those lacking HML α and HMR a cryptic mating loci. Furthermore, we transplanted the synthetic yeast chromosome V to build a haploid polysynthetic chromosome strain by using this method together with an endoreduplication intercross strategy. The CRISPR/Cas9 mating-type switching method will be useful in building the complete synthetic yeast (Sc2.0) genome. Importantly, it is a generally useful method to build polyploids of a defined genotype and generally expedites strain construction, for example, in the construction of fully a/a /α/α isogenic tetraploids., (Copyright © 2018 Xie et al.)

Microbial production of monoterpenes is often limited by their cytotoxicity and in vivo conversion. Therefore, alleviating cytotoxicity and reducing conversion by chassis engineering are highly desirable. On the other hand, engineering key enzymes is also critical for improving monoterpenes production through facilitating the biosynthesis process. Here we critically review recent advances in cytotoxicity alleviation, reducing in vivo conversion, selecting geranyl diphosphate synthase and engineering monoterpene synthases. These achievements would lead to the development of superior chassis with improved tolerance to cytotoxicity and rationally tailored metabolites profiles to improve titer, yield and productivity for the production of monoterpenes by microbial cells., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Combinatorial design is an effective strategy to acquire the optimal solution in complex systems. In this study, the combined effects of pathway combination, promoters' strength fine-tuning, copy numbers and integration locus variations caused by δ-integration were explored in Saccharomyces cerevisiae using geranylgeraniol (GGOH) production as an example. Two GGOH biosynthetic pathway branches were constructed. In branch 1, GGOH was converted from isopentenyl pyrophosphate (IPP) and farnesyl diphosphate (FPP). In branch 2, GGOH was derived directly from IPP and dimethylallyl pyrophosphate (DMAPP). Regulated by 10 combinations of 11 diverse promoters, a fusion gene BTS1-ERG20, a heterologous geranylgeranyl diphosphate synthase from Sulfolobus acidocaldarius (GGPPSsa) and an endogenous N-terminal truncated gene 3-hydroxyl-3-methylglutaryl-CoA reductase isoenzyme 1 (tHMGR), were incorporated into yeast by δ-integration, leading to a series of GGOH producing strains with yields ranging from 18.45 mg/L to 161.82 mg/L. The yield was further increased to 437.52 mg/L by optimizing the fermentation medium. Consequently, the GGOH yield reached 1315.44 mg/L in a 5-L fermenter under carbon restriction strategy. Our study not only opens large opportunities for downstream diterpenes overproductions, but also demonstrates that pathway optimization based on combinatorial design is a promising strategy to engineer microbes for overproducing natural products with complex structure.

Peripheral inflammation induces sensitization of nociceptive spinal cord neurons. Both spinal tumor necrosis factor (TNF) and neuronal membrane insertion of Ca 2+ permeable AMPA receptor (AMPAr) contribute to spinal sensitization and resultant pain behavior, molecular mechanisms connecting these two events have not been studied in detail. Intrathecal (i.t.) injection of TNF-blockers attenuated paw carrageenan-induced mechanical and thermal hypersensitivity. Levels of GluA1 and GluA4 from dorsal spinal membrane fractions increased in carrageenan-injected rats compared to controls. In the same tissue, GluA2 levels were not altered. Inflammation-induced increases in membrane GluA1 were prevented by i.t. pre-treatment with antagonists to TNF, PI3K, PKA and NMDA. Interestingly, administration of TNF or PI3K inhibitors followed by carrageenan caused a marked reduction in plasma membrane GluA2 levels, despite the fact that membrane GluA2 levels were stable following inhibitor administration in the absence of carrageenan. TNF pre-incubation induced increased numbers of Co 2+ labeled dorsal horn neurons, indicating more neurons with Ca 2+ permeable AMPAr. In parallel to Western blot results, this increase was blocked by antagonism of PI3K and PKA. In addition, spinal slices from GluA1 transgenic mice, which had a single alanine replacement at GluA1 ser 845 or ser 831 that prevented phosphorylation, were resistant to TNF-induced increases in Co 2+ labeling. However, behavioral responses following intraplantar carrageenan and formalin in the mutant mice were no different from littermate controls, suggesting a more complex regulation of nociception. Co-localization of GluA1, GluA2 and GluA4 with synaptophysin on identified spinoparabrachial neurons and their relative ratios were used to assess inflammation-induced trafficking of AMPAr to synapses. Inflammation induced an increase in synaptic GluA1, but not GluA2. Although total GluA4 also increased with inflammation, co-localization of GluA4 with synaptophysin, fell short of significance. Taken together these data suggest that peripheral inflammation induces a PI3K and PKA dependent TNFR1 activated pathway that culminates with trafficking of calcium permeable AMPAr into synapses of nociceptive dorsal horn projection neurons., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Under normal condition, there are a few lipid droplets in skeletal muscle. But in skeletal muscle acute injury, muscular dystrophy, muscle atrophy, obesity, diabetes and other pathological conditions, the fat deposition in skeletal muscle increases, which implicate that the fat deposition may play an important role in the pathogenesis of these diseases. However, the mechanisms of development and regulation of fat deposition in skeletal muscle are not clear. Clarifying the key signaling pathways and regulatory factors that affect fat deposition in skeletal muscle, and exploring new ways to improve the fat deposition in skeletal muscle will not only help to deepen our understanding of the pathogenesis of these diseases, but also provide new ideas for the treatment of these diseases. This paper reviews the research progresses and main mechanisms of fat deposition in skeletal muscle.

The efficacy of epidermal growth factor receptor- targeted therapy is significantly associated with Kirsten rat sarcoma viral oncogene homolog (KRAS) and B-raf serine/threonine kinase proto-oncogene (BRAF) mutation in patients with colorectal cancer (CRC), for which the standard gene testing is currently performed using tumor tissue DNA. The aim of the present study was to compare the presence of KRAS and BRAF mutations in the serum exosome and primary tumor tissue from patients with CRC. Genomic DNA were extracted from the tumor tissues of 35 patients with histologically-confirmed CRC and exosomal mRNA were obtained from peripheral blood, which were collected from the corresponding patients prior to surgery. Three mutations in the KRAS gene (codons 12, 13 and 61) and a mutation in the BRAF gene (codon 600) were detected using a polymerase chain reaction-based sequencing method and their presence were compared between tumor tissues and the matched serum exosomes. The KRAS mutation rates in tumor tissues and the matched serum exosomes were 57.6 and 42.4%, respectively, which was not significantly different (P=0.063). The detection rate of the BRAF mutation was 24.2 and 18.2% in tumor tissues and the matched serum exosomes, respectively, and there was no significant difference (P=0.500). The patients with CRC that had a KRAS mutation of codon 12 in exon 2 in their tumor tissues and serum exosomes were significantly older compared with those without this mutation (tumor tissue, P=0.002; serum exosome, P=0.022). The sensitivity of KRAS and BRAF mutation detection using exosomal mRNA was 73.7 and 75%, respectively. The specificity of the detected mutations exhibited an efficiency of 100%, and the total consistency rate was 94.9 and 93.9% for KRAS and BRAF mutations, respectively. These results suggested that serum exosomal mRNA may be used as a novel source for the rapid and non-invasive genotyping of patients with CRC.

Manipulation of monoterpene synthases to maximize flux towards targeted products from GPP (geranyl diphosphate) is the main challenge for heterologous monoterpene overproduction, in addition to cell toxicity from compounds themselves. In our study, by manipulation of the key enzymes geraniol synthase (GES) and farnesyl diphosphate synthase (Erg20), geraniol (a valuable acyclic monoterpene alcohol) overproduction was achieved in Saccharomyces cerevisiae with truncated 3-hydroxy-3-methylglutaryl-coenzyme reductase (tHMGR) and isopentenyl diphosphate isomerase (IDI1) overexpressed. The expressions of all above engineered genes were under the control of Gal promoter for alleviating product toxicity. Geraniol production varied from trace amount to 43.19mg/L (CrGES, GES from Catharanthus roseus) by screening of nine GESs from diverse species. Further through protein structure analysis and site-directed mutation in CrGES, it was firstly demonstrated that among the high-conserved amino acid residues located in active pocket, Y436 and D501 with strong affinity to diphosphate function group, were critical for the dephosphorylation (the core step for geraniol formation). Moreover, the truncation position of the transit peptide from the N-terminus of CrGES was found to influence protein expression and activity significantly, obtaining a titer of 191.61mg/L geraniol in strain with CrGES truncated at S43 (t3CrGES). Furthermore, directed by surface electrostatics distribution of t3CrGES and Erg20 WW (Erg20 F96W-N127W ), co-expression of the reverse fusion of Erg20 ww /t3CrGES and another copy of Erg20 WW promoted the geraniol titer to 523.96mg/L at shakes flask level, due to enhancing GPP accessibility led by protein interaction of t3CrGES-Erg20 WW and the free Erg20 WW . Eventually, a highest reported titer of 1.68g/L geraniol in eukaryote cells was achieved in 2.0L fed-batch fermentation under carbon restriction strategy. Our research opens large opportunities for other microbial production of monoterpenes. It also sets a good reference for desired compounds overproduction in microorganisms in terms of manipulation of key enzymes by protein engineering and metabolic engineering., (Copyright © 2017. Published by Elsevier Inc.)

Background: The prognostic value of latent membrane protein 1 (LMP1) in non-Hodgkin lymphoma (NHL) has been evaluated in several studies. However, the conclusions remain controversial., Methods: We searched relevant literatures from Embase, PubMed, and China National Knowledge Infrastructure Platform databases and performed a meta-analysis to evaluate the prognostic significance of LMP1 expression in NHL. Pooled hazard ratio (HR), 95% confidence interval (CI), and P value were calculated. Nine relevant studies were analyzed in this meta-analysis. We performed a pooled analysis to assess the association between LMP1 expression and overall survival of NHL patients., Results: Our results revealed that LMP1-positive NHL patients had significantly poorer outcomes than LMP1-negative patients (HR = 2.13, 95% CI = 1.31-3.46, Pheterogeneity = 0.005, I = 63.5%). Furthermore, in the subgroup analysis stratified by country, a statistically significant association was found among Chinese (HR = 2.80, 95% CI = 1.53-5.15, Pheterogeneity = 0.342, I = 6.9%); however, no statistically significant relations were found among Japanese (HR = 1.55, 95% CI = 0.74-3.24, Pheterogeneity = 0.020, I = 65.7%)., Conclusion: The expression of LMP1 can be considered a poor predictor of survival in patients with NHL. In addition, LMP1 expression assessment could provide more detailed information for patients with NHL and could be used to optimize therapeutic schemes.

Debugging a genome sequence is imperative for successfully building a synthetic genome. As part of the effort to build a designer eukaryotic genome, yeast synthetic chromosome X (synX), designed as 707,459 base pairs, was synthesized chemically. SynX exhibited good fitness under a wide variety of conditions. A highly efficient mapping strategy called pooled PCRTag mapping (PoPM), which can be generalized to any watermarked synthetic chromosome, was developed to identify genetic alterations that affect cell fitness ("bugs"). A series of bugs were corrected that included a large region bearing complex amplifications, a growth defect mapping to a recoded sequence in FIP1 , and a loxPsym site affecting promoter function of ATP2 PoPM is a powerful tool for synthetic yeast genome debugging and an efficient strategy for phenotype-genotype mapping., (Copyright © 2017, American Association for the Advancement of Science.)