Exploring the molecular mechanisms of Lrp/AsnC-type transcription regulator DecR, an L-cysteine-responsive feast/famine regulatory protein.

The Lrp/AsnC family of transcriptional regulators is commonly found in prokaryotes and is associated with the regulation of amino acid metabolism. However, it remains unclear how the L-cysteine-responsive Lrp/AsnC family regulator perceives and responds to L-cysteine. Here, we try to elucidate the molecular mechanism of the L-cysteine-responsive transcriptional regulator. Through 5'RACE and EMSA, we discovered a 15 bp incompletely complementary pair palindromic sequence essential for DecR binding, which differed slightly from the binding sequence of other Lrp/AsnC transcription regulators. Using alanine scanning, we identified the L-cysteine binding site on DecR and found that different Lrp/AsnC regulators adjust their binding pocket's side-chain residues to accommodate their specific effector. MD simulations were then conducted to explore how ligand binding influences the allosteric behavior of the protein. PCA and in silico docking revealed that ligand binding induced perturbations in the linker region, triggering conformational alterations and leading to the relocalization of the DNA-binding domains, enabling the embedding of the DNA-binding region of DecR into the DNA molecule, thereby enhancing DNA-binding affinity. Our findings can broaden the understanding of the recognition and regulatory mechanisms of the Lrp/AsnC-type transcription factors, providing a theoretical basis for further investigating the molecular mechanisms of other transcription factors.

Engineering of the Lrp/AsnC-type transcriptional regulator DecR as a genetically encoded biosensor for multilevel optimization of L-cysteine biosynthesis pathway in Escherichia coli.

L-cysteine is an important sulfur-containing amino acid being difficult to produce by microbial fermentation. Due to the lack of high-throughput screening methods, existing genetically engineered bacteria have been developed by simply optimizing the expression of L-cysteine-related genes one by one. To overcome this limitation, in this study, a biosensor-based approach for multilevel biosynthetic pathway optimization of L-cysteine from the DecR regulator variant of Escherichia coli was applied. Through protein engineering, we obtained the DecRN29Y/C81E/M90Q/M99E variant-based biosensor with improved specificity and an 8.71-fold increase in dynamic range. Using the developed biosensor, we performed high-throughput screening of the constructed promoter and RBS combination library, and successfully obtained the optimized strain, which resulted in a 6.29-fold increase in L-cysteine production. Molecular dynamics (MD) simulations and electrophoretic mobility shift analysis (EMSA) showed that the N29Y/C81E/M90Q/M99E variant had enhanced induction activity. This enhancement may be due to the increased binding of the variant to DNA in the presence of L-cysteine, which enhances transcriptional activation. Overall, our biosensor-based strategy provides a promising approach for optimizing biosynthetic pathways at multiple levels. The successful implementation of this strategy demonstrates its potential for screening improved recombinant strains.

A Cadaveric Study of the Optimal Isometric Region on the Anterolateral Surface of the Knee in Anterolateral Ligament Reconstruction.

OBJECTIVE: Isolated intra-articular anterior cruciate ligament (ACL) reconstruction is not capable of restoring instability in many cases leading some to recommend concomitant anterolateral ligament (ALL) reconstruction. The satisfactory fixation site and graft length change are crucial in ligament reconstruction to restore the ALL function and avoid some unwanted graft behavior. The purpose of this investigation is to determine the optimal isometric region on the anterolateral aspect of the knee for ALL reconstruction using a three-dimensional optical instrument and a suture similar to an intraoperative isometric test. METHODS: Six freshly frozen cadaveric human knees were used in this study. Data regarding the anterolateral surface were obtained using an optical measurement system to create a three-dimensional model. Nine points were selected on the femur (F1-F9) and tibia (Ta-Ti) respectively. The three-dimensional length change between each pair of tibial and femoral points was measured during passive knee flexion from 0° to 90° in 15° increments. Subsequently, five femoral points (A-E) were selected from the lateral femur, located in different areas relative to the lateral femoral epicondyle, and three tibial reference points (T1-T3) were selected in the isometric test. The changes in the length between each pair of reference points were measured using sutures. The 95% confidence interval for the rate of length change was estimated using the mean and standard deviation of the maximum rate of length change at different flexion angles, and the data were expressed as the mean (95% confidence interval) and compared with the maximum acceptable rate of change (10%). RESULTS: The maximum acceptable change rate for ligament reconstruction is 10%, and the mean maximum rates and the 95% confidence interval (CI) of length change for the point combinations were calculated. Among all the combined points measured using the optical measurement system and the suture, the qualified point combination for reconstruction was F3 (8mm posterior and 8mm proximal to the lateral femoral epicondyle)-Tb (8mm proximal to the midpoint between the center of Gerdy's tubercle and the fibula head), A (posterior and proximal to the lateral femoral epicondyle)-T2 (10mm below the joint line)and A-T3 (15 mm below the joint line). The position of F3-Tb and A-T2 are close to each other. CONCLUSION: The most isometric area of the femur for ALL reconstruction was posterior and proximal to the lateral femoral epicondyle. We recommend that the initial location of the femoral point be set at 8 mm posterior and 8 mm proximal to the lateral femoral epicondyle and the tibial point at approximately 10 mm below the joint line, midway between Gerdy's tubercle and fibular head, and subsequently adjusted to the most satisfactory position according to the isometric test.

Prognosis Prediction of Lung Adenocarcinoma Patients Based on Molecular Subgroups of DNA Methylation.

Lung adenocarcinoma (LUAD) is a malignant tumor with high mortality. At present, the clinicopathologic feature is the main breakthrough to assess the prognosis of LUAD patients. However, in most cases, the results are less than satisfactory. Cox regression analysis was conducted in this study to obtain methylation sites with significant prognostic relevance based on mRNA expression, DNA methylation data, and clinical data of LUAD from The Cancer Genome Atlas Program database. LUAD patients were grouped into 4 subtypes according to different methylation levels using K-means consensus cluster analysis. By survival analysis, patients were grouped into high-methylation and low-methylation groups. Later, 895 differentially expressed genes (DEGs) were obtained. Eight optimal methylation signature genes associated with prognosis were screened by Cox regression analysis, and a risk assessment model was constructed based on these genes. Samples were then classified into high-risk and low-risk groups depending on the risk assessment model, and prognostic, predictive ability was assessed using survival and receiver operating characteristic (ROC) curves. The results showed that this risk model had a great efficacy in predicting the prognosis of patients, and it was, therefore, able to be an independent prognostic factor. At last, the enrichment analysis demonstrated that the signaling pathways, including cell cycle, homologous recombination, P53 signaling pathway, DNA replication, pentose phosphate pathway, and glycolysis gluconeogenesis were remarkably activated in the high-risk group. In general, we construct an 8-gene model based on DNA methylation molecular subtypes by a series of bioinformatics methods, which can provide new insights for predicting the prognosis of patients with LUAD.

A Modified Arthroscopic Outside-in Shoulder Release Approach for Severe Shoulder Stiffness.

OBJECTIVE: Arthroscopic release is effective for patients with shoulder stiffness, but the traditional inside-out procedure cannot effectively alleviate the mobility of some severe stiff shoulder and even cause itrogenic injuries sometimes. The aim of this study is to evaluate the clinical efficacy and advantages of a modified outside-in shoulder release approach for severe shoulder stiffness. METHODS: Included in this retrospective study were 15 patients (five male and 10 female) with severe shoulder stiffness who underwent modified outside-in shoulder release surgery at our hospital between June 2019 and March 2021. Of them, 10 patients had a primary frozen shoulder and five had secondary shoulder stiffness, involving the right shoulder in six cases and the left shoulder in nine cases. The mean age of the 15 patients was 56.7 (34-69) years. The patients were instructed to exercise passively from second-day post-operation and enhance the rehabilitation exercise gradually. All patients received a range of motion (ROM) examination before and after surgery. The American Shoulder and Elbow Surgeon's Score (ASES), Constant Score (CS), and Visual Analog Scale (VAS) score for pain were recorded. All data were tested by normal distribution first and then by paired T test, otherwise by Wilcoxon rank sum test. RESULTS: The mean follow-up period was 18.2 (12-33) months. Compared with the preoperative value, the mean ASES score at the final follow-up improved from 38.4 ± 7.37 to 88.13 ± 6.33 points; the mean CS score from 43.27 ± 6.71 to 78.74 ± 6.93 points; the mean VAS score from 5.07 ± 1.03 to 0.81 ± 0.83 points; forward flexion from 81.93° ± 11.45° to 156.73° ± 9.12°; abduction from 65.93° ± 16.82° to 144.80° ± 8.83°; neutral external rotation from 13.53° ± 10.38° to 51.20° ± 4.77°; internal rotation from the buttock to waist (L3), all showing a significant difference (P < 0.0001). No serious complication was observed in any patient during the postoperative follow-up periods. CONCLUSION: The present study has demonstrated that the modified arthroscopic outside-in shoulder release approach can improve ROM of patients and alleviate pain effectively, proving it to be an appropriate surgical option for the treatment of severe shoulder stiffness.

Highly efficient ethylene production via electrocatalytic hydrogenation of acetylene under mild conditions.

Renewable energy-based electrocatalytic hydrogenation of acetylene to ethylene (E-HAE) under mild conditions is an attractive substitution to the conventional energy-intensive industrial process, but is challenging due to its low Faradaic efficiency caused by competitive hydrogen evolution reaction. Herein, we report a highly efficient and selective E-HAE process at room temperature and ambient pressure over the Cu catalyst. A high Faradaic efficiency of 83.2% for ethylene with a current density of 29 mA cm-2 is reached at -0.6 V vs. the reversible hydrogen electrode. In-situ spectroscopic characterizations combined with first-principles calculations reveal that electron transfer from the Cu surface to adsorbed acetylene induces preferential adsorption and hydrogenation of the acetylene over hydrogen formation, thus enabling a highly selective E-HAE process through the electron-coupled proton transfer mechanism. This work presents a feasible route for high-efficiency ethylene production from E-HAE.

Controllable CO adsorption determines ethylene and methane productions from CO2 electroreduction.

Among all CO2 electroreduction products, methane (CH4) and ethylene (C2H4) are two typical and valuable hydrocarbon products which are formed in two different pathways: hydrogenation and dimerization reactions of the same CO intermediate. Theoretical studies show that the adsorption configurations of CO intermediate determine the reaction pathways towards CH4/C2H4. However, it is challenging to experimentally control the CO adsorption configurations at the catalyst surface, and thus the hydrocarbon selectivity is still limited. Herein, we seek to synthesize two well-defined copper nanocatalysts with controllable surface structures. The two model catalysts exhibit a high hydrocarbon selectivity toward either CH4 (83%) or C2H4 (93%) under identical reduction conditions. Scanning transmission electron microscopy and X-ray absorption spectroscopy characterizations reveal the low-coordination Cu0 sites and local Cu0/Cu+ sites of the two catalysts, respectively. CO-temperature programed desorption, in-situ attenuated total reflection Fourier transform infrared spectroscopy and density functional theory studies unveil that the bridge-adsorbed CO (COB) on the low-coordination Cu0 sites is apt to be hydrogenated to CH4, whereas the bridge-adsorbed CO plus linear-adsorbed CO (COB + COL) on the local Cu0/Cu+ sites are apt to be coupled to C2H4. Our findings pave a new way to design catalysts with controllable CO adsorption configurations for high hydrocarbon product selectivity.

Inhibition of Smad3 promotes the healing of rotator cuff injury in a rat model.

To investigate the effect of inhibiting transforming growth factor-ß (TGF-ß1)/Smad2/3 signaling on rotator cuff (RC) healing. A bilateral supraspinatus tendon detachment-repair model of Sprague-Dawley (SD) rats was utilized. A total of 120 SD rats were randomly assigned to six groups and each group received the subacromial injection of normal saline, empty vectors, or lentiviral vectors containing small interfering RNA against TGF-ß1, Smad2, Smad3 at the bone-tendon junction. Biomechanical and histological analyses were performed to evaluate bone-tendon junction healing quality at 8 weeks after repair. Histologically, scar healing was found in all surgical groups. Animals with inhibited Smad3 exhibited better bone-tendon junction structures with higher density, parallel orientation, and collagen fiber continuity than other surgical group animals. Immunohistochemistry revealed that the protein expression level of collagen I in animals with inhibited Smad3 was more prominent compared with all other surgical groups. Biomechanically, Animals with inhibited Smad3 showed better results in the maximum load at 4, 6, and 8 weeks after surgery compared with other surgical groups. Besides, C3H10T1/2 (Smad3-) cells increased TT-D6 cell migration and tendon-associated genes expression (scleraxis, tenascin C, collagen I) in coculture system. We conclude that inhibition of Smad3 promotes RC tendon healing in the rat supraspinatus model.

Early Follow-Up of Arthroscopic Latarjet Procedure with Screw or Suture-Button Fixation for Recurrent Anterior Shoulder Instability.

OBJECTIVE: To evaluate the early clinical and radiographic results of arthroscopic Latarjet procedure using screw or suture-button fixation in patients with recurrent anterior shoulder dislocation. METHODS: Twelve patients who underwent arthroscopic Latarjet procedure between January 2015 and December 2018 at our institution were retrospectively studied. Data of the patients' history, including age, gender, side of affected arm, body mass index (BMI), and the number of dislocations since fist dislocation were collected. Preoperative and postoperative clinical follow-up data were evaluated using Walch-Duplay score, American Shoulder and Elbow Society (ASES) score, and modified Rowe score. Active external rotation and active internal rotation at 90° of abduction as well as active elevation were evaluated preoperatively and postoperatively. The position and healing condition of the transferred coracoid bony graft were also assessed using computed tomography (CT) and Mimics 19.0 software. RESULTS: Mean follow-up was 24.9 months (range, 13 to 53 months) of all patients. At final follow-up, the average ASES score (preoperative vs postoperative values) had improved from 68.9 ± 7.9 to 91.1 ± 6.1 in screw fixation group and 68.9 ± 8.9 to 87.5 ± 6.7 in suture-button fixation group; the average Rowe score (preoperative vs postoperative values) had improved from 25.0 ± 8.4 to 92.5 ± 4.2 in screw fixation group and 21.7 ± 13.7 to 93.3 ± 4.1 in suture-button fixation group; the average of Walch-Duplay score (preoperative vs postoperative values) had improved from 12.5 ± 15.1 to 91.7 ± 4.1 in screw fixation group and 18.3 ± 20.7 to 88.3 ± 7.5 in button fixation group. The forward flexion was 175.0° ± 8.4° preoperatively and 178.3° ± 4.1° postoperatively in screw fixation group while 174.8° ± 10.2° preoperatively and 175.0° ± 5.5° postoperatively in suture-button fixation group. The active external rotation was 77.5° ± 5.2° preoperatively and 71.7° ± 4.1° postoperatively in screw fixation group while 72.5° ± 6.9° preoperatively and 68.3° ± 7.5° postoperatively in suture-button fixation group. The average of active internal rotation was 66.7° ± 6.1° preoperatively and 67.5° ± 6.1° postoperatively in screw fixation group while 68.3° ± 11.3° preoperatively and 66.7° ± 7.5° postoperatively in suture-button fixation group. In postoperative CT scan, 91.7% grafts midline center were located at or under the equator in the en face view; 75% of the bone blocks were flush to the glenoid face in the axial view, with only two grafts exhibiting slight medial placement in screw fixation group (33.3%) and one graft exhibiting slight lateral placement in suture-button fixation group (16.7%). All grafts achieved bone union. Graft absorption mostly occurred outside of the "best-fit" circle. The average bony absorption rates of the coracoid grafts were 25.2% and 10.18% in screw fixation group and suture-button fixation group, respectively, at 6 months postoperative follow-up. CONCLUSION: Both suture-button fixation and screw fixation techniques in arthroscopic Latarjet procedure revealed excellent clinical outcomes with low complication rates in the early follow-up. The suture-button fixation exhibited a flexible fixation pattern that allowed for self-correction to some extent, even slight lateralization could finally remodel over time.

Kartogenin enhances the therapeutic effect of bone marrow mesenchymal stem cells derived exosomes in cartilage repair.

The effectiveness of mesenchymal stem cells (MSC) in the treatment of cartilage diseases has been demonstrated to be attributed to the paracrine mechanisms, especially the mediation of exosomes. But the exosomes derived from unsynchronized MSCs may be nonhomogeneous and the therapeutic effect varies between samples. Aim: To produce homogeneous and more effective exosomes for the regeneration of cartilage. Materials & methods: In this study we produced specific exosomes from bone marrow MSCs (BMSC) through kartogenin (KGN) preconditioning and investigated their performance in either in vitro or in vivo experiments. Results & conclusion: The exosomes derived from KGN-preconditioned BMSCs (KGN-BMSC-Exos) performed more effectively than the exosomes derived from BMSCs (BMSC-Exos). KGN preconditioning endowed BMSC-Exos with stronger chondral matrix formation and less degradation.

Application of assisted portal under anterior horn of lateral meniscus for the treatment of discoid meniscus injury.

PURPOSE: The assisted inferior anterolateral portal under anterior horn of the lateral meniscus (UAHLM portal) was applied to treat the lateral discoid meniscus injury conveniently and the clinical outcomes were evaluated. METHODS: A retrospective review was conducted on 60 patients who underwent arthroscopic surgery with a symptomatic discoid lateral meniscus. Normal anterolateral/anteromedial portals assisted with UAHLM portal (1-2 cm inferior to the anterolateral portal) were used. All patients were followed up for 24-48 months (median, 33 months) and evaluated by MRI images and clinical outcomes including clinical findings, Lysholm scores and IKDC scores. RESULTS: After meniscus plasty with or without repair, most of the upper layer of lateral meniscuses was retained. A total of 54 patients (16 males and 38 females, 42 ±â€¯17.8 years old) showed satisfactory clinical results without requiring reoperation after a median follow-up time of 33 months. At final follow-up, a full range of motion was achieved in all patients. MRI indicated the thickness of anterior horn of lateral meniscus was (5.38 ±â€¯1.09 mm) before the operation and (4.04 ±â€¯0.71 mm) after the operation at the 2-year follow-up; clinical outcomes were improved significantly than the baseline: positive McMurray test (50 vs. 2, P< 0.001), Lysholm score (64.9 ±â€¯9.0 vs. 94.7 ±â€¯4.9, P< 0.001), and IKDC score (54.4 ±â€¯7.7 vs. 92.6 ±â€¯4.3, P< 0.001). No significant complication was observed during the follow-up. CONCLUSION: Thus, this technique with assisted UAHLM portal was convenient for arthroscopic discoid meniscus plasty and meniscus repair and served as an effective method in patients with a symptomatic discoid lateral.

Glycol chitosan/oxidized hyaluronic acid hydrogels functionalized with cartilage extracellular matrix particles and incorporating BMSCs for cartilage repair.

In this article, we fabricated a bioactive hydrogel composed of glycol chitosan (G-CS) and oxidized hyaluronic acid (OHA) via Schiff base reaction. Cartilage extracellular matrix (ECM) particles with different concentrations were used to functionalize G-CS/OHA (S1) hydrogel. The results demonstrated that S3 (G-CS/OHA/ECM 2% w/v) hydrogel exhibited the most suitable compression strength and provided the optimal environment for proliferation of bone marrow mesenchymal stem cells (BMSCs). To assess the chondroinductivity of ECM in vitro, we compared the chondrogenesis of BMSCs in S1 (G-CS/OHA) and S3 (G-CS/OHA/ECM 2% w/v) hydrogels. The results confirmed that the higher levels of glycosaminoglycans (GAGs) and collagen type II (COL II) were accumulated in S3 hydrogel. In vivo, cartilage defects of rats generated most mature tissue within BMSCs-laden S3 hydrogel (S3/BMSCs group). The tissues were more integrative and contained higher levels of COL II and GAGs compared to the other groups. All these results suggested that the G-CS/OHA hydrogel functionalized with ECM particles is a good candidate biomaterial to be applied in cartilage tissue engineering.

Promoting Ethylene Selectivity from CO2 Electroreduction on CuO Supported onto CO2 Capture Materials.

Cu is a unique catalyst for CO2 electroreduction, since it can catalyze CO2 reduction to a series of hydrocarbons, alcohols, and carboxylic acids. Nevertheless, such Cu catalysts suffer from poor selectivity. High pressure of CO2 is considered to facilitate the activity and selectivity of CO2 reduction. Herein, a new strategy is presented for CO2 reduction with improved C2 H4 selectivity on a Cu catalyst by using CO2 capture materials as the support at ambient pressure. N-doped carbon (Nx C) was synthesized through high-temperature carbonization of melamine and l-lysine. We observed that the CO2 uptake capacity of Nx C depends on both the microporous area and the content of pyridinic N species, which can be controlled by the carbonization temperature (600-800 °C). The as-prepared CuO/Nx C catalysts exhibit a considerably higher C2 H4 faradaic efficiency (36 %) than CuO supported on XC-72 carbon black (19 %), or unsupported CuO (20 %). Moreover, there is a good linear relationship between the C2 H4 faradaic efficiency and CO2 uptake capacity of the supports for CuO. The local high CO2 concentration near Cu catalysts, created by CO2 capture materials, was proposed to increase the coverage of CO intermediate, which is favorable for the coupling of two CO units in the formation of C2 H4 . This study demonstrates that pairing Cu catalysts with CO2 capture supports is a promising approach for designing highly effective CO2 reduction electrocatalysts.

Kartogenin Enhanced Chondrogenesis in Cocultures of Chondrocytes and Bone Mesenchymal Stem Cells.

Articular cartilage has poor capability of regeneration due to the avascular surrounding and low metabolic activity. Kartogenin (KGN), an emerging nonprotein heterocyclic compound, was screened to stimulate chondrogenic differentiation of bone mesenchymal stem cells (BMSCs). Coculturing BMSCs and chondrocytes was reported to overcome the shortcomings of forming fibroblastic and hypertrophic cartilages. In this study, KGN was incorporated into the Col-Tgel hydrogel to form a Gel/Cell/KGN complex, which fabricated an appropriate microenvironment for effective cartilage regeneration of BMSCs and/or chondrocytes. The complexes that incorporated KGN, BMSCs, and chondrocytes achieved higher lubricin expression and extracellular matrix production, such as characteristic glycosaminoglycans (GAGs) and collagen type II (COL II), compared to the monocultures of BMSCs or chondrocytes in vitro. The complexes compounding KGN, BMSCs, and chondrocytes (at an optimal ratio in the in vitro experiment) were transplanted into rat models to evaluate the repair effects. Our results suggested that the interaction between BMSCs and chondrocytes can substitute the use of growth factors to some degree and indicated the role of KGN in chondrogenesis induction. Besides, it is the first time (to our knowledge) that the expression of lubricin was found to be delayed in the coculture of mixed cells comparing with GAGs and COL II, which could be significant in cartilage tissue engineering.

Antitumor activity of a recombinant soluble ectodomain of mutant human fibroblast growth factor receptor-2 IIIc.

The fibroblast growth factor (FGF) signaling pathway is a recognized target of cancer therapy. We have developed a strong inhibitor (S252W mutant soluble ectodomain of FGF recptor-2 IIIc, msFGFR2) that binds FGFs and blocks the activation of FGFRs. Thermodynamic binding studies indicated that msFGFR2 bound FGF-2 16.9 times as strongly as wild-type soluble FGFR2IIIc ectodomain (wsFGFR2). It successfully suppressed the growth, angiogenesis, and metastasis of two tumor cell lines in vitro and in vivo, and it potently inhibited cancer cell proliferation but not normal cell proliferation. Therefore, msFGFR2 is a useful probe for FGF-dependent signaling pathways and a potential broad-spectrum antitumor agent.