Cell-free Protein Synthesis System for Building Synthetic Cells.

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for the core machinery of the Central Dogma, standing as an optimal chassis for the integration and assembly of diverse artificial cellular mimicry systems. Despite its frequent use in the fabrication of synthetic cells, establishing a tailored and robust CFPS system for a specific application remains a nontrivial challenge. In this methods paper, we present a comprehensive protocol for the CFPS system, routinely employed in constructing synthetic cells. This protocol encompasses key stages in the preparation of the CFPS system, including the cell extract, template preparation, and routine expression optimization utilizing a fluorescent reporter protein. Additionally, we show representative results by encapsulating the CFPS system within various micro-compartments, such as monolayer droplets, double-emulsion vesicles, and chambers situated atop supported lipid bilayers. Finally, we elucidate the critical steps and conditions necessary for the successful assembly of these CFPS systems in distinct environments. We expect that our approach will facilitate the establishment of good working practices among various laboratories within the continuously expanding synthetic cell community, thereby accelerating progress in the field of synthetic cell development.

Causal roles of gut microbiota in cholangiocarcinoma etiology suggested by genetic study.

BACKGROUND: Cholangiocarcinoma (CCA) is a highly malignant biliary tract cancer with poor prognosis. Previous studies have implicated the gut microbiota in CCA, but evidence for causal mechanisms is lacking. AIM: To investigate the causal relationship between gut microbiota and CCA risk. METHODS: We performed a two-sample mendelian randomization study to evaluate potential causal associations between gut microbiota and CCA risk using genome-wide association study summary statistics for 196 gut microbial taxa and CCA. Genetic variants were used as instrumental variables. Multiple sensitivity analyses assessed result robustness. RESULTS: Fifteen gut microbial taxa showed significant causal associations with CCA risk. Higher genetically predicted abundance of genus Eubacteriumnodatum group, genus Ruminococcustorques group, genus Coprococcus, genus Dorea, and phylum Actinobacteria were associated with reduced risk of gallbladder cancer and extrahepatic CCA. Increased intrahepatic CCA risk was associated with higher abundance of family Veillonellaceae, genus Alistipes, order Enterobacteriales, and phylum Firmicutes. Protective effects against CCA were suggested for genus Collinsella, genus Eisenbergiella, genus Anaerostipes, genus Paraprevotella, genus Parasutterella, and phylum Verrucomicrobia. Sensitivity analyses indicated these findings were reliable without pleiotropy. CONCLUSION: This pioneering study provides novel evidence that specific gut microbiota may play causal roles in CCA risk. Further experimental validation of these candidate microbes is warranted to consolidate causality and mechanisms.

Genome-wide systematic survey and analysis of the RNA helicase gene family and their response to abiotic stress in sweetpotato.

Sweetpotato (Ipomoea batatas (L.) Lam.) holds a crucial position as one of the staple foods globally, however, its yields are frequently impacted by environmental stresses. In the realm of plant evolution and the response to abiotic stress, the RNA helicase family assumes a significant role. Despite this importance, a comprehensive understanding of the RNA helicase gene family in sweetpotato has been lacking. Therefore, we conducted a comprehensive genome-wide analysis of the sweetpotato RNA helicase family, encompassing aspects such as chromosome distribution, promoter elements, and motif compositions. This study aims to shed light on the intricate mechanisms underlying the stress responses and evolutionary adaptations in sweetpotato, thereby facilitating the development of strategies for enhancing its resilience and productivity. 300 RNA helicase genes were identified in sweetpotato and categorized into three subfamilies, namely IbDEAD, IbDEAH and IbDExDH. The collinearity relationship between the sweetpotato RNA helicase gene and 8 related homologous genes from other species was explored, providing a reliable foundation for further study of the sweetpotato RNA helicase gene family's evolution. Furthermore, through RNA-Seq analysis and qRT-PCR verification, it was observed that the expression of eight RNA helicase genes exhibited significant responsiveness to four abiotic stresses (cold, drought, heat, and salt) across various tissues of ten different sweetpotato varieties. Sweetpotato transgenic lines overexpressing the RNA helicase gene IbDExDH96 were generated using A.rhizogenes-mediated technology. This approach allowed for the preliminary investigation of the role of sweetpotato RNA helicase genes in the response to cold stress. Notably, the promoters of RNA helicase genes contained numerous cis-acting elements associated with temperature, hormone, and light response, highlighting their crucial role in sweetpotato abiotic stress response.

Association Between Different Metabolic Markers and Metabolic Syndrome in an Outpatient Hyperuricemia Population: A Cross-Sectional Study.

Background: Hyperuricemia (HUA) is often associated with metabolic syndrome (MetS). However, the role of different metabolic markers in the screening of MetS in out-patients with hyperuricemia is unclear. The study aims to investigate the relationship between different metabolic indexes and MetS. Methods: A total of 399 hyperuricemia patients from Tianjin Medical University General Hospital from 2022 to 2023 were included in this study. We collected demographic, anthropometric, and biochemical data on each subject. And calculate serum uric acid-to-creatinine ratio (SUA/Cr), serum uric acid to high-density lipoprotein cholesterol (UHR), triglyceride to high-density lipoprotein cholesterol (THR), and triglyceride and glucose (TyG) index. Binary logistic regression modeling was performed to explore the association between different metabolic markers and MetS. Results: Out of the 399 subjects, 28.3% had MetS. UHR, THR, and TyG index were significantly higher in the MetS group than those in the control group (p<0.05), which were associated with an increased risk of MetS. However, SUA/Cr was not associated with MetS (p>0.05). TyG index had a stronger relationship with the risk of MetS (OR 5.476, 95% CI 2.210-13.569, p<0.001). The cut-off with the biggest Youden index of the TyG index was 9.13 and the area under the curve (AUC) was 0.719. Conclusion: A high TyG index is associated with an increased risk of MetS in HUA patients. These findings might help screen MetS in individuals with HUA and could be more standardized management of patients with high uric acid in outpatient clinics.

Bottom-Up Synthetic Biology Using Cell-Free Protein Synthesis.

Technical advances in biotechnology have greatly accelerated the development of bottom-up synthetic biology. Unlike top-down approaches, bottom-up synthetic biology focuses on the construction of a minimal cell from scratch and the application of these principles to solve challenges. Cell-free protein synthesis (CFPS) systems provide minimal machinery for transcription and translation, from either a fractionated cell lysate or individual purified protein elements, thus speeding up the development of synthetic cell projects. In this review, we trace the history of the cell-free technique back to the first in vitro fermentation experiment using yeast cell lysate. Furthermore, we summarized progresses of individual cell mimicry modules, such as compartmentalization, gene expression regulation, energy regeneration and metabolism, growth and division, communication, and motility. Finally, current challenges and future perspectives on the field are outlined.

Aquaporins and CO2 diffusion across biological membrane.

Despite the physiological significance of effective CO2 diffusion across biological membranes, the underlying mechanism behind this process is not yet resolved. Particularly debatable is the existence of CO2-permeable aquaporins. The lipophilic characteristic of CO2 should, according to Overton's rule, result in a rapid flux across lipid bilayers. However, experimental evidence of limited membrane permeability poses a challenge to this idea of free diffusion. In this review, we summarized recent progress with regard to CO2 diffusion, and discussed the physiological effects of altered aquaporin expression, the molecular mechanisms of CO2 transport via aquaporins, and the function of sterols and other membrane proteins in CO2 permeability. In addition, we highlight the existing limits in measuring CO2 permeability and end up with perspectives on resolving such argument either by determining the atomic resolution structure of CO2 permeable aquaporins or by developing new methods for measuring permeability.

Advancing synthetic biology through cell-free protein synthesis.

The rapid development of synthetic biology has enabled the production of compounds with revolutionary improvements in biotechnology. DNA manipulation tools have expedited the engineering of cellular systems for this purpose. Nonetheless, the inherent constraints of cellular systems persist, imposing an upper limit on mass and energy conversion efficiencies. Cell-free protein synthesis (CFPS) has demonstrated its potential to overcome these inherent constraints and has been instrumental in the further advancement of synthetic biology. Via the removal of the cell membranes and redundant parts of cells, CFPS has provided flexibility in directly dissecting and manipulating the Central Dogma with rapid feedback. This mini-review summarizes recent achievements of the CFPS technique and its application to a wide range of synthetic biology projects, such as minimal cell assembly, metabolic engineering, and recombinant protein production for therapeutics, as well as biosensor development for in vitro diagnostics. In addition, current challenges and future perspectives in developing a generalized cell-free synthetic biology are outlined.

Applications and prospects of cryo-EM in drug discovery.

Drug discovery is a crucial part of human healthcare and has dramatically benefited human lifespan and life quality in recent centuries, however, it is usually time- and effort-consuming. Structural biology has been demonstrated as a powerful tool to accelerate drug development. Among different techniques, cryo-electron microscopy (cryo-EM) is emerging as the mainstream of structure determination of biomacromolecules in the past decade and has received increasing attention from the pharmaceutical industry. Although cryo-EM still has limitations in resolution, speed and throughput, a growing number of innovative drugs are being developed with the help of cryo-EM. Here, we aim to provide an overview of how cryo-EM techniques are applied to facilitate drug discovery. The development and typical workflow of cryo-EM technique will be briefly introduced, followed by its specific applications in structure-based drug design, fragment-based drug discovery, proteolysis targeting chimeras, antibody drug development and drug repurposing. Besides cryo-EM, drug discovery innovation usually involves other state-of-the-art techniques such as artificial intelligence (AI), which is increasingly active in diverse areas. The combination of cryo-EM and AI provides an opportunity to minimize limitations of cryo-EM such as automation, throughput and interpretation of medium-resolution maps, and tends to be the new direction of future development of cryo-EM. The rapid development of cryo-EM will make it as an indispensable part of modern drug discovery.

Reconstitution of a Reversible Membrane Switch via Prenylation by One-Pot Cell-Free Expression.

Reversible membrane targeting of proteins is one of the key regulators of cellular interaction networks, for example, for signaling and polarization. So-called "membrane switches" are thus highly attractive targets for the design of minimal cells but have so far been tricky to reconstitute in vitro. Here, we introduce cell-free prenylated protein synthesis (CFpPS), which enables the synthesis and membrane targeting of proteins in a single reaction mix including the prenylation machinery. CFpPS can confer membrane affinity to any protein via addition of a 4-peptide motif to its C-terminus and offers robust production of prenylated proteins not only in their soluble forms but also in the direct vicinity of biomimetic membranes. Thus, CFpPS enabled us to reconstitute the prenylated polarity hub Cdc42 and its regulatory protein in vitro, implementing a key membrane switch. We propose CFpPS to be a versatile and effective platform for engineering complex features, such as polarity induction, in synthetic cells.

Molecular Mechanisms of Mercury-Sensitive Aquaporins.

Aquaporins are transmembrane channels that allow for the passive permeation of water and other small molecules across biological membranes. Their channel activities are sensitive to mercury ions. Intriguingly, while most aquaporins are inhibited by mercury ions, several aquaporins are activated by mercury ions. The molecular basis of the opposing aquaporin regulation by mercury remains poorly understood. Herein, we investigated AqpZ inhibition and AQP6 activation upon binding of mercury ions using solid-state NMR (ssNMR) and molecular dynamics (MD) simulations. Based on the structure of the Hg-AqpZ complex constructed by MD simulations and ssNMR, we identified that the pore closure was caused by mercury-induced conformational changes of the key residue R189 in the selectivity filter region, while pore opening was caused by conformational changes of residues H181 and R196 in the selectivity filter region in AQP6. Both conformational changes were caused by the disruption of the H-bond network of R189/R196 by mercury. The molecular details provided a structural basis for mercury-mediated functional changes in aquaporins.

Microbial degradation and valorization of poly(ethylene terephthalate) (PET) monomers.

The polyethylene terephthalate (PET) is one of the major plastics with a huge annual production. Alongside with its mass production and wide applications, PET pollution is threatening and damaging the environment and human health. Although mechanical or chemical methods can deal with PET, the process suffers from high cost and the hydrolyzed monomers will cause secondary pollution. Discovery of plastic-degrading microbes and the corresponding enzymes emerges new hope to cope with this issue. Combined with synthetic biology and metabolic engineering, microbial cell factories not only provide a promising approach to degrade PET, but also enable the conversion of its monomers, ethylene glycol (EG) and terephthalic acid (TPA), into value-added compounds. In this way, PET wastes can be handled in environment-friendly and more potentially cost-effective processes. While PET hydrolases have been extensively reviewed, this review focuses on the microbes and metabolic pathways for the degradation of PET monomers. In addition, recent advances in the biotransformation of TPA and EG into value-added compounds are discussed in detail.

The Arabidopsis thaliana trehalose-6-phosphate phosphatase gene AtTPPI regulates primary root growth and lateral root elongation.

Roots are the main organs through which plants absorb water and nutrients. As the key phytohormone involved in root growth, auxin functions in plant environmental responses by modulating auxin synthesis, distribution and polar transport. The Arabidopsis thaliana trehalose-6-phosphate phosphatase gene AtTPPI can improve root architecture, and tppi1 mutants have significantly shortened primary roots. However, the mechanism underlying the short roots of the tppi1 mutant and the upstream signaling pathway and downstream genes regulated by AtTPPI are unclear. Here, we demonstrated that the AtTPPI gene could promote auxin accumulation in AtTPPI-overexpressing plants. By comparing the transcriptomic data of tppi1 and wild-type roots, we found several upregulations of auxin-related genes, including GH3.3, GH3.9 and GH3.12, may play an important role in the AtTPPI gene-mediated auxin transport signaling pathway, ultimately leading to changes in auxin content and primary root length. Moreover, increased AtTPPI expression can regulate primary root growth and lateral root elongation under different concentration of nitrate conditions. Overall, constitutive expression of AtTPPI increased auxin contents and improved lateral root elongation, constituting a new method for improving the nitrogen utilization efficiency of plants.

Research progress in the role of distal femoral valgus cut angle in accurate total knee arthroplasty / 中华创伤杂志

The distal femoral fracture, tibial plateau fracture and patellar fracture may all develop into traumatic knee arthritis, which is probably associated with knee dysfunction problems. Total knee arthroplasty (TKA) is an effective treatment for end-stage knee arthritis. The distal femoral valgus cut angle (VCA) is an important reference for distal femoral resection in TKA and significantly affects postoperative prosthesis position and lower extremity alignment after TKA. For VCA, the specific methods for definition, measurement methods, clinical application and influencing factors are currently controversial. Hence, the authors review the research progress in the role of VCA in TKA from the above-mentioned four aspects, hoping to provide a reference for accurate preoperative planning and intraoperative performance of TKA.

Application and research progress of intelligent accuracy technological aids in total knee arthroplasty / 中华创伤杂志

Total knee arthroplasty (TKA) is an effective treatment for end-stage knee disease, with the postoperative alignment, component position, soft tissue balance, and prosthesis matching being key factors for the success of TKA. In order to achieve more accurate postoperative alignment and component position, better soft tissue balance and prosthesis matching for longer prosthesis longevity, better postoperative function and higher patient satisfaction, various intelligent accuracy technological aids such as computer assisted navigation (CAN), patient specific instrumentation (PSI), surgical robots, microsensors, customized implants (CI) and personalized 3D preoperative planning have emerged and are given high expectation. In this paper, the authors review the application and research progress of the above technological aids mainly from aspects of alignment, component position, clinical outcomes and cost analysis, so as to provide a reference for the application of related technological aids in TKA.

Impact of chronic obstructive pulmonary disease on postoperative complications and short-term prognosis in patients undergoing oesophagectomy / 中国胸心血管外科临床杂志

@#Objective    To provide clinical reference for the perioperative management of esophageal cancer patients with different stages of chronic obstructive pulmonary disease (COPD) through investigating the impact of COPD on postoperative complications and survival in esophageal cancer patients undergoing oesophagectomy. Methods    The clinical data of 163 patients who underwent radical resection of esophageal cancer in our department from January 2015 to January 2018 were retrospectively analyzed, including 124 males and 39 females, with a median age of 64 years (IQR: 23.8 years). They were divided into a COPD group (n=87) and a non-COPD group (n=76) according to the presence of COPD before operation. The clinical data were collected and the postoperative complications and 2-year survival between the two groups were compared and analyzed. Results    The incidence of major postoperative complications (pulmonary infection, respiratory failure, arrhythmia and anastomotic leakage) in the COPD group were higher than those in the non-COPD group (all P<0.05). Spearman correlation analysis showed that the severity of preoperative COPD was positively correlated with the incidence of postoperative complications in patients with esophageal cancer (r=0.437, P<0.001). The incidence of postoperative respiratory failure and mortality in patients with severe COPD were significantly higher than those in patients without COPD and those with mild or moderate COPD. The 2-year survival rate of patients with esophageal cancer in the COPD group was lower than that in the non-COPD group (56.1%vs. 78.5.%, P=0.001), and the severity of COPD was negatively correlated to the survival rate. Conclusion    COPD significantly increases the incidence of postoperative complications in patients with esophageal cancer, which is not conducive to the prognosis of patients, and the severity of COPD is correlated with postoperative complications and 2-year survival rate.

Low-Concentration C2 H6 Capture Enabled by Size Matching in the Ultramicropore.

Low-concentration ethane capture is crucial for environmental protection and natural gas purification. The ideal physisorbent with strong C2 H6 interaction and large C2 H6 uptake at low-concentration level has rarely been reported, due to the large pKa value and small quadrupole moment of C2 H6 . Herein, we demonstrate the perfectly size matching between the ultramicropore (pore size of 4.6 Å) and ethane (kinetic diameter of 4.4 Å) in a nickel pyridine-4-carboxylate metal-organic framework (IISERP-MOF2), which enables the record-breaking performance for low concentration C2 H6 capture. IISERP-MOF2 exhibits the large C2 H6 adsorption enthalpy of 56.7 kJ/mol, and record-high C2 H6 uptake at low pressure of 0.01-0.1 bar and 298 K (1.8 mmol/g at 0.01 bar). Molecule simulations and C2 H6 -loading crystal structure analysis revealed that the maximized interaction sites in IISERP-MOF2 with ethane molecule originates the strong C2 H6 adsorption. The dynamic breakthrough experiments for gas mixtures of C2 H6 /N2 (1/999, v/v) and C2 H6 /CH4 (5/95, v/v) proved the excellent low-concentration C2 H6 capture performance.

Clinical Correlation Between Overactive Bladder and Allergy in Children.

BACKGROUND: The International Children's Continence Society defines overactive bladder (OAB) as a clinical syndrome characterized by urgency of urination usually accompanied by frequent urination and nocturia symptoms. This study aims to explore the correlation between overactive bladder (OAB) and allergy in children. METHOD: The clinical characteristics of 918 patients diagnosed with OAB from January 2020 to March 2021 were retrospectively analyzed. Risk factors for OAB were analyzed using logistic regression analysis, and the effect of desloratadine in the treatment of OAB was evaluated. RESULTS: The incidence of allergic cough or allergic rhinitis in the mild OAB group was higher than the moderate-severe group. Urodynamics demonstrated that the proportion of patients with a sensitive bladder in the overactive detrusor group was significantly higher than the non-overactive detrusor group. The effective rate of treatment of OAB in patients complicated with allergies and taking desloratadine was 90.14%, which was significantly higher than in patients who were not taking desloratadine, and blood IgE level was a risk factor of ineffective treatment with desloratadine. CONCLUSION: OAB is correlated with allergies in children, and desloratadine can effectively improve OAB symptoms.

[Quality evaluation of Gastrodiae Rhizoma standard decoction based on UPLC fingerprint and quantitative analysis multi-components method].

To establish the quantitative analysis multi-components with a single-marker(QAMS) method for six components and fingerprint of standard decoction of Gastrodiae Rhizoma, verify the accuracy and feasibility of the method, and evaluate the quality of standard decoction. Based on UPLC with gastrodin as the internal standard, relative correction factors of p-hydroxybenzyl alcohol, parishin E, parishin B, parishin C, parishin A and gastrodin were determined by investigating the column temperature, flow rate, chromatographic columns and multi-point concentration correction. The total contents in 18 batches of standard decoction of Gastrodiae Rhizoma and the similarity were determined to calculate the similarity. The results of standard curve method, external standard one-point method and quantitative analysis multi-components with a single-marker(QAMS) were compared, and the results showed that there was no significant difference among these three methods. By analyzing the results of standard decoctions from different origins, it can be seen that the quality of Gastrodia standard decoctions derived from Anhui and Yunnan was better, followed by Shaanxi and Hubei, and relatively poor in Gansu, with similarities all above 0.90 in the fingerprints. Therefore, the QAMS method that can measure the contents of gastrodin, p-hydroxybenzyl alcohol, parishin E, parishin B, parishin C and parishin A in standard decoction of Gastrodiae Rhizoma combined with fingerprint is accurate, feasible and fast, which can be used to evaluate the quality of standard decoction of Gastrodiae Rhizoma, and also provide a reference for the research on the quality standards of raw materials for Gastrodiae Rhizoma prepared slices and alike.

Functional Analysis of Aquaporin Water Permeability Using an Escherichia coli-Based Cell-Free Protein Synthesis System.

Aquaporins are essential water channel proteins found in all kingdoms of life. Although the water permeability of aquaporins has been well characterized, sample preparation for aquaporin water permeability assays remains challenging and time-consuming. Besides the difficulty in overexpressing membrane proteins in a cell-based expression system, the unique requirement for homogeneity in aquaporin proteoliposome sample preparations for water transport assays further increases the complexity. In this study, a complementary Cell-free Protein Synthesis (CFPS) method is described in detail, providing three different strategies for the preparation of aquaporin proteoliposome samples. Aquaporin can be produced either as a pellet fraction and then resolubilized, or co-translationally as a detergent-soluble fraction. Furthermore, aquaporin can be directly incorporated into liposomes, which was included in the CFPS reactions. Although proteoliposomes tend to fuse during the incubation of the CFPS reactions, an additional treatment of the fused samples with detergent, followed by a detergent removal step, can re-form homogenously sized proteoliposomes suitable for functional analysis. Using this method, we successfully characterized aquaporins from both prokaryotic and eukaryotic organisms. In particular, in the presence of liposomes, the developed CFPS expression system is a fast and convenient method for sample preparation for the functional analysis of aquaporins.

Impact of Maternal Selenium Supplementation from Late Gestation and Lactation on Piglet Immune Function.

The present work aimed at assessing passive, innate, and acquired immunity in piglets from sows supplemented with either organic or inorganic selenium (Se). A total of 12 multiparous pregnant sows were randomly allocated to three groups: selenium-deficient, corn and soy-based diet base diet (BD), 0.3 mg Se/kg as hydroxy-selenomethionine (OH-SeMet), and 0.3 mg Se/kg as sodium selenite (SS). The feeding trial was carried out from gd 84 to weaning on postpartum day 21 (ppd 21). On gd 98 and 105, sows were vaccinated with hen egg white lysozyme (HEWL) to assess passive immunity. On ppd 23, weaned piglets were intramuscularly challenged with lipopolysaccharide (LPS) to trigger an acute-phase response. On ppd 14, 28, and 35, piglets were vaccinated with ovalbumin (OVA) to assess OVA-specific immunoglobulin G (IgG) and dermal hypersensitivity responses. Se levels in piglet plasma, muscle, and liver on ppd 21 were higher in OH-SeMet group. On ppd 2, piglet HEWL-specific IgG levels in OH-SeMet group were significantly increased. IL-10 and haptoglobin (HP) levels in OH-SeMet group were significantly increased 2 h and 48 h post-LPS simulation, respectively. The OVA-specific IgG levels in BD group were significantly higher than the other two groups, and the IL-4 concentration following whole blood ex vivo challenge with either OVA or mitogen was significantly increased in OH-SeMet group. OVA-specific skin swelling was lower in OH-SeMet and SS groups at 3 h and 6 h. This suggests that sow supplementation with OH-SeMet enhances mainly passive immunity through IgG maternal transfer and can influence piglet innate and acquired immunity.