Intracellular biosensor-based dynamic regulation to manipulate gene expression at the spatiotemporal level.

The use of intracellular, biosensor-based dynamic regulation strategies to regulate and improve the production of useful compounds have progressed significantly over previous decades. By employing such an approach, it is possible to simultaneously realize high productivity and optimum growth states. However, industrial fermentation conditions contain a mixture of high- and low-performance non-genetic variants, as well as young and aged cells at all growth phases. Such significant individual variations would hinder the precise controlling of metabolic flux at the single-cell level to achieve high productivity at the macroscopic population level. Intracellular biosensors, as the regulatory centers of metabolic networks, can real-time sense intra- and extracellular conditions and, thus, could be synthetically adapted to balance the biomass formation and overproduction of compounds by individual cells. Herein, we highlight advances in the designing and engineering approaches to intracellular biosensors. Then, the spatiotemporal properties of biosensors associated with the distribution of inducers are compared. Also discussed is the use of such biosensors to dynamically control the cellular metabolic flux. Such biosensors could achieve single-cell regulation or collective regulation goals, depending on whether or not the inducer distribution is only intracellular.

A systematic review and meta-analysis comparing outcomes following total knee arthroplasty for rheumatoid arthritis versus for osteoarthritis.

PURPOSE: Total knee arthroplasty (TKA) in patients with osteoarthritis (OA) are considered to be a successful procedure, but with little being known about outcomes in patients with rheumatoid arthritis (RA). The aim of this study was to compare the outcomes of TKA in patients with RA versus OA. METHODS: Data were obtained from PubMed, Cochrane Library, EBSCO and Scopus for all available studies comparing the outcomes of THA in RA and OA patients (From January 1, 2000 to October 15, 2022). Outcomes of interest included infection, revision, venous thromboembolism (VTE), mortality, periprosthetic fractures, prosthetic loosening, length of stay, and satisfaction. Two reviewers independently assessed each study for quality and extracted data. The quality of the studies was scored using the Newcastle-Ottawa scale (NOS). RESULTS: Twenty-four articles with a total 8,033,554 patients were included in this review. The results found strong evidence for increased risk of overall infection (OR = 1.61, 95% CI, 1.24-2.07; P = 0.0003), deep infection (OR = 2.06, 95% CI, 1.37-3.09; P = 0.0005), VTE (OR = 0.76, 95% CI, 0.61-0.93; P = 0.008), pulmonary embolism (PE) (OR = 0.84, 95% CI, 0.78-0.90; P<0.00001), periprosthetic fractures (OR = 1.87, 95% CI, 1.60-2.17; P<0.00001); and reasonable evidence for increased risk of deep venous thrombosis (DVT) (OR = 0.74, 95% CI, 0.54-0.99; P = 0.05), and length of stay (OR = 0.07, 95% CI, 0.01-0.14; P = 0.03) after TKA in patients with RA versus OA. There were no significant differences in superficial site infection (OR = 0.84,95% CI, 0.47-1.52; P = 0.57), revision (OR = 1.33,95% CI, 0.79-2.23; P = 0.28), mortality (OR = 1.16,95% CI, 0.87-1.55; P = 0.32), and prosthetic loosening (OR = 1.75, 95% CI, 0.56-5.48; P = 0.34) between the groups. CONCLUSION: Our study demonstrated that patients with RA have a higher risk of postoperative infection, VTE, periprosthetic fracture, and lengths of stay, but did not increase revision rate, prosthetic loosening and mortality compared to patients with OA following TKA. In conclusion, despite RA increased incidence of postoperative complications, TKA should continue to be presented as an effective surgical procedure for patients whose conditions are intractable to conservative and medical management of RA.

Clinical effects of interspace between the popliteal artery and capsule of the posterior knee block with multimodal analgesia for total knee arthroplasty: a systematic review and meta-analysis.

PURPOSE: Combination of regional anaesthesia technique that is most effective in analgesia and postoperative functional outcome with the fewest complications needs investigation. Interspace between the popliteal artery and the capsule of the posterior knee block (IPACK) has been introduced clinically. We evaluated the efficacy of IPACK in combination with other nerve blocks after total knee arthroplasty. METHODS: Data were obtained from PubMed, Cochrane Library, Web of Science, and Sciencedirect. Studies that compared outcomes using IPACK combined with other regional nerve blocks after total knee arthroplasty with other analgesic modalities and those which used pain scores or opioid consumption as primary or secondary outcomes were included. RESULTS: Seventeen articles (20 trials, 1652 patients) were included. IPACK supplementation significantly reduced rest pain scores after total knee arthroplasty at postoperative hours 8-12(95%CI - 0.85 [- 1.36, - 0.34], I2 = 94%, p = 0.001), postoperative day 1 (95% CI - 0.49 [- 0.85, - 0.14], I2 = 87%, p = 0.006), and postoperative day 2 (95% CI - 0.28 [- 0.51, -0.05], I2 = 72%, p = 0.02); there was no significant difference at postoperative day 3 or discharge (95% CI - 0.14 [- 0.33, 0.05], I2 = 0%, p = 0.14). Combination treatment resulted in reduced dynamic pain scores at postoperative hours 8-12 (95%CI - 0.52 [- 0.92, - 0.12], I2 = 86%, p = 0.01) and postoperative day 1(95% CI - 0.49 [- 0.87, - 0.11], I2 = 88%, p = 0.01). There was no difference between postoperative day 2(95% CI - 0.29 [- 0.63, 0.05], I2 = 80%, p = 0.09), postoperative day 3 or discharge (95% CI - 0.45 [- 0.92, 0.02], I2 = 83%, p = 0.06). In addition, it strongly reduced postoperative opioid consumption within 24 H (95% CI - 0.76 [- 1.13, - 0.39], I2 = 85%, p < 0.00001), 24-48 H (95% CI - 0.43 [- 0.85, - 0.01], I2 = 83%, p = 0.04), and total opioid use (95% CI - 0.64 [- 1.07, - 0.22], I2 = 86%, p = 0.003). Although IPACK supplementation improved timed up and go test and walking distance at postoperative day 2, there was no statistically significant difference at other time periods or obvious improvement in knee range of motion and quadriceps strength. IPACK block supplementation could shorten the length of stay (LOS) (95% CI - 0.40 [- 0.64, - 0.15], I2 = 70%, p = 0.001) and improve patient satisfaction (95% CI 0.43 [0.01, 0.84], I2 = 87%, p = 0.04). CONCLUSION: Based on these results, IPACK supplementation, in addition to standard postoperative analgesia, can be used effectively and safely to relieve early postoperative pain after total knee arthroplasty.

Claisen Condensation Reaction Mediated Pimelate Biosynthesis via the Reverse Adipate-Degradation Pathway and Its Isoenzymes.

Pimelic acid is an important seven-carbon dicarboxylic acid, which is broadly applied in various fields. The industrial production of pimelic acid is mainly through a chemical method, which is complicated and environmentally unfriendly. Herein, we found that pimelic acid could be biosynthesized by the reverse adipate-degradation pathway (RADP), a typical Claisen condensation reaction that could be applied to the arrangement of C-C bond. In order to strengthen the supply of glutaryl-CoA precursor, PA5530 protein was used to transport glutaric acid. Subsequently, we discovered that the enzymes in the BIOZ pathway are isoenzyme of the RADP pathway enzymes. By combining the isoenzymes of the two pathways, the titer of pimelic acid reached 36.7 mg ⋅ L-1 under the optimal combination, which was increased by 382.9 % compared with the control strain B-3. It was also the highest titer of pimelic acid biosynthesized by Claisen condensation reaction, laying the foundation for the production of pimelic acid and its derivatives.

Programmable cross-ribosome-binding sites to fine-tune the dynamic range of transcription factor-based biosensor.

Currently, predictive translation tuning of regulatory elements to the desired output of transcription factor (TF)-based biosensors remains a challenge. The gene expression of a biosensor system must exhibit appropriate translation intensity, which is controlled by the ribosome-binding site (RBS), to achieve fine-tuning of its dynamic range (i.e. fold change in gene expression between the presence and absence of inducer) by adjusting the translation level of the TF and reporter. However, existing TF-based biosensors generally suffer from unpredictable dynamic range. Here, we elucidated the connections and partial mechanisms between RBS, translation level, protein folding and dynamic range, and presented a design platform that predictably tuned the dynamic range of biosensors based on deep learning of large datasets cross-RBSs (cRBSs). In doing so, a library containing 7053 designed cRBSs was divided into five sub-libraries through fluorescence-activated cell sorting to establish a classification model based on convolutional neural network in deep learning. Finally, the present work exhibited a powerful platform to enable predictable translation tuning of RBS to the dynamic range of biosensors.

Development of a growth coupled and multi-layered dynamic regulation network balancing malonyl-CoA node to enhance (2S)-naringenin biosynthesis in Escherichia coli.

Metabolic heterogeneity and dynamic changes in metabolic fluxes are two inherent characteristics of microbial fermentation that limit the precise control of metabolisms, often leading to impaired cell growth and low productivity. Dynamic metabolic engineering addresses these challenges through the design of multi-layered and multi-genetic dynamic regulation network (DRN) that allow a single cell to autonomously adjust metabolic flux in response to its growth and metabolite accumulation conditions. Here, we developed a growth coupled NCOMB (Naringenin-Coumaric acid-Malonyl-CoA-Balanced) DRN with systematic optimization of (2S)-naringenin and p-coumaric acid-responsive regulation pathways for real-time control of intracellular supply of malonyl-CoA. In this scenario, the acyl carrier protein was used as a novel critical node for fine-tuning malonyl-CoA consumption instead of direct repression of fatty acid synthase commonly employed in previous studies. To do so, we first engineered a multi-layered DRN enabling single cells to concurrently regulate acpH, acpS, acpT, acs, and ACC in malonyl-CoA catabolic and anabolic pathways. Next, the NCOMB DRN was optimized to enhance the synergies between different dynamic regulation layers via a biosensor-based directed evolution strategy. Finally, a high producer obtained from NCOMB DRN approach yielded a 8.7-fold improvement in (2S)-naringenin production (523.7 ± 51.8 mg/L) with a concomitant 20% increase in cell growth compared to the base strain using static strain engineering approach, thus demonstrating the high efficiency of this system for improving pathway production.

Biosensor-Based Multigene Pathway Optimization for Enhancing the Production of Glycolate.

Glycolate is widely used in industry, especially in the fields of chemical cleaning, cosmetics, and medical materials, and has broad market prospects for the future. Recent advances in metabolic engineering and synthetic biology have significantly improved the titer and yield of glycolate. However, an expensive inducer was used in previous studies, which is not feasible for use in large-scale industrial fermentations. To constitutively biosynthesize glycolate, the expression level of each gene of the glycolate synthetic pathway needs to be systemically optimized. The main challenge of multigene pathway optimization is being able to select or screen the optimum strain from the randomly assembled library by an efficient high-throughput method within a short time. To overcome these challenges, we firstly established a glycolate-responsive biosensor and developed agar plate- and 48-well deep-well plate-scale high-throughput screening methods for the rapid screening of superior glycolate producers from a large library. A total of 22 gradient-strength promoter-5'-untranslated region (UTR) complexes were randomly cloned upstream of the genes of the glycolate synthetic pathway, generating a large random assembled library. After rounds of screening, the optimum strain was obtained from 6 × 105 transformants in a week, and it achieved a titer of 40.9 ± 3.7 g/liter glycolate in a 5-liter bioreactor. Furthermore, high expression levels of the enzymes YcdW and GltA were found to promote glycolate production, whereas AceA has no obvious impact on glycolate production. Overall, the glycolate biosensor-based pathway optimization strategy presented in this work provides a paradigm for other multigene pathway optimizations. IMPORTANCE The use of strong promoters, such as pTrc and T7, to control gene expression not only needs the addition of expensive inducers but also results in excessive protein expression that may result in unbalanced metabolic flux and the waste of cellular building blocks and energy. To balance the metabolic flux of glycolate biosynthesis, the expression level of each gene needs to be systemically optimized in a constitutive manner. However, the lack of high-throughput screening methods restricted glycolate synthetic pathway optimization. Our work firstly established a glycolate-response biosensor, and agar plate- and 48-well plate-scale high-throughput screening methods were then developed for the rapid screening of optimum pathways from a large library. Finally, we obtained a glycolate-producing strain with good biosynthetic performance, and the use of the expensive inducer isopropyl-ß-d-thiogalactopyranoside (IPTG) was avoided, which broadens our understanding of the mechanism of glycolate synthesis.

Highly Efficient Ir-CoOx Hybrid Nanostructures for the Selective Hydrogenation of Furfural to Furfuryl Alcohol.

Decoration of noble metals with transition-metal oxides has been intensively studied for heterogeneous catalysis. However, controllable syntheses of metal-metal oxide heterostructures are difficult, and elucidation of such interfaces is still challenging. In this work, supported IrCo alloy nanoparticles are transformed into supported Ir-CoOx close-contact nanostructures by in situ calcination and following selective reduction. Relative to Ir/Al2O3, Ir-CoOx/Al2O3 shows greatly enhanced activities for the hydrogenation of furfural derivatives to the corresponding furfuryl alcohol derivatives with more than 99% selectivity and demonstrates significantly improved activities and selectivity for hydrogenations of α,ß-unsaturated aldehydes to α,ß-unsaturated alcohols. The modification of Ir surfaces with CoOx prevents Ir nanoparticles from growing, achieving high thermal and catalytic stabilities. Theoretic calculation suggests that the better catalytic performance of Ir-CoOx/Al2O3 is ascribed to the Ir-CoOx interaction, which promotes the absorption of furfural as well as desorption of furfuryl alcohol, resulting in enhanced catalytic activities.

The 3-ketoacyl-CoA thiolase: an engineered enzyme for carbon chain elongation of chemical compounds.

Because of their function of catalyzing the rearrangement of the carbon chains, thiolases have attracted increasing attentions over the past decades. The 3-ketoacyl-CoA thiolase (KAT) is a member of the thiolase, which is capable of catalyzing the Claisen condensation reaction between the two acyl-CoAs, thereby achieving carbon chain elongation. In this way, diverse value-added compounds might be synthesized starting from simple small CoA thioesters. However, most KATs are hampered by low stability and poor substrate specificity, which has hindered the development of large-scale biosynthesis. In this review, the common characteristics in the three-dimensional structure of KATs from different sources are summarized. Moreover, structure-guided rational engineering is discussed as a strategy for enhancing the performance of KATs. Finally, we reviewed the metabolic engineering applications of KATs for producing various energy-storage molecules, such as n-butanol, fatty acids, dicarboxylic acids, and polyhydroxyalkanoates. KEY POINTS: • Summarize the structural characteristics and catalyzation mechanisms of KATs. • Review on the rational engineering to enhance the performance of KATs. • Discuss the applications of KATs for producing energy-storage molecules.

Computer-aided engineering of adipyl-CoA synthetase for enhancing adipic acid synthesis.

OBJECTIVE: To enhance adipic acid production, a computer-aided approach was employed to engineer the adipyl-CoA synthetase from Thermobifida fusca by combining sequence analysis, protein structure modeling, in silico site-directed mutagenesis, and molecular dynamics simulation. RESULTS: Two single mutants of T. fusca adipyl-CoA synthetase, E210ßN and E210ßQ, achieved a specific enzyme activity of 1.95 and 1.84 U/mg, respectively, which compared favorably with the 1.48 U/mg for the wild-type. The laboratory-level fermentation experiments showed that E210ßN and E210ßQ achieved a maximum adipic acid titer of 0.32 and 0.3 g/L. In contrast, the wild-type enzyme yielded a titer of 0.15 g/L under the same conditions. Molecular dynamics (MD) simulations revealed that the mutants (E210ßN and E210ßQ) could accelerate the dephosphorylation process in catalysis and enhance enzyme activity. CONCLUSIONS: The combined computational-experimental approach provides an effective strategy for enhancing enzymatic characteristics, and the mutants may find a useful application for producing adipic acid.

Fine-tuning the (2S)-naringenin synthetic pathway using an iterative high-throughput balancing strategy.

Metabolic engineering consistently demands to produce the maximum carbon and energy flux to target chemicals. To balance metabolic flux, gene expression levels of artificially synthesized pathways usually fine-tuned using multimodular optimization strategy. However, forward construction is an engineering conundrum because a vast number of possible pathway combinations need to be constructed and analyzed. Here, an iterative high-throughput balancing (IHTB) strategy was established to thoroughly fine-tune the (2S)-naringenin biosynthetic pathway. A series of gradient constitutive promoters from Escherichia coli were randomly cloned upstream of pathway genes, and the resulting library was screened using an ultraviolet spectrophotometry-fluorescence spectrophotometry high-throughput method, which was established based on the interactions between AlCl3 and (2S)-naringenin. The metabolic flux of the screened high-titer strains was analyzed and iterative rounds of screening were performed based on the analysis results. After several rounds, the metabolic flux of the (2S)-naringenin synthetic pathway was balanced, reaching a final titer of 191.9 mg/L with 29.2 mg/L p-coumaric acid accumulation. Chalcone synthase was speculated to be the rate-limiting enzyme because its expression level was closely related to the production of both (2S)-naringenin and p-coumaric acid. The established IHTB strategy can be used to efficiently balance multigene pathways, which will accelerate the development of efficient recombinant strains.

Controlled Synthesis and Enhanced Catalytic Activity of Well-Defined Close-Contact Pd-ZnO Nanostructures.

In this study, PdZn-ordered intermetallic nanoparticles (NPs) were prepared in liquid phase by butyllithium co-reduction of their precursors at 240 °C. Through calcination and subsequent reduction with H2, the synthesized PdZn NPs were then in situ transformed into Pd-ZnO heteroaggregate nanocatalysts on alumina supports. Various characterization techniques, such as diffuse reflectance Fourier transform infrared with CO probes, transmission electron microscopy, X-ray diffraction, H2 temperature-programmed reduction, and X-ray photoelectron spectra, reveal that PdZn NPs are ordered intermetallic compounds, and in situ transformation of PdZn alloy NPs results in close-contact Pd-ZnO heteroaggregates, where the interfaces are highly active and the interaction between Pd and ZnO prevents the active particles from agglomeration. The catalytic hydrogenations of nitrophenols over Pd/Al2O3 and Pd-ZnO/Al2O3 were investigated. The results show that Pd-ZnO/Al2O3 illustrates an enhanced catalytic activity relative to Pd/Al2O3, and no obvious activity degradation was observed in the recycle catalytic experiments over such nanostructures. It is concluded that the Pd-ZnO interaction not only enhances the catalytic hydrogenation activity but also promotes the thermal and catalytic stability.

Metabolic engineering of Escherichia coli BL21 (DE3) for de novo production of L-DOPA from D-glucose.

BACKGROUND: Production of L-tyrosine is gaining grounds as the market size of 3,4-dihydroxyphenyl-L-alanine (L-DOPA) is expected to increase due to increasing cases of Parkinson's disease a neurodegenerative disease. Attempts to overproduce L-tyrosine for conversion to L-DOPA has stemmed on the overexpressing of critical pathway enzymes, an introduction of feedback-resistant enzymes, and deregulation of transcriptional regulators. RESULTS: An E. coli BL21 (DE3) was engineered by deleting tyrR, ptsG, crr, pheA and pykF while directing carbon flow through the overexpressing of galP and glk. TktA and PpsA were also overexpressed to enhance the accumulation of E4P and PEP. Directed evolution was then applied on HpaB to optimize its activity. Three mutants, G883R, G883A, L1231M, were identified to have improved activity as compared to the wild-type hpaB showing a 3.03-, 2.9- and 2.56-fold increase in L-DOPA production respectively. The use of strain LP-8 resulted in the production of 691.24 mg/L and 25.53 g/L of L-DOPA in shake flask and 5 L bioreactor, respectively. CONCLUSION: Deletion of key enzymes to channel flux towards the shikimate pathway coupled with the overexpression of pathway enzymes enhanced the availability of L-tyrosine for L-DOPA production. Enhancing the activity of HpaB increased L-DOPA production from glucose and glycerol. This work demonstrates that increasing the availability of L-tyrosine and enhancing enzyme activity ensures maximum L-DOPA productivity.

Proximal femoral nail anti-rotation versus cementless bipolar hemiarthroplasty for unstable femoral intertrochanteric fracture in the elderly: a retrospective study.

BACKGROUND: The treatment for unstable intertrochanteric fractures in the elderly has always been a controversial issue. The aim in this study was to compare the curative effects of proximal femoral nail anti-rotation (PFNA) and cementless bipolar hemiarthroplasty (CPH) on femoral intertrochanteric fracture in the elderly. METHODS: From March 2008 to December 2012, 108 elderly patients with femoral intertrochanteric fractures were treated by PFNA or CPH. There were 63 males and 45 females, aged 75.3-99.1 years [(83.7 ± 5.6) years]. The patients' bone mineral density was routinely measured, and the fractures were classified according to Evans-Jensen. The patients were divided into CPH group and PFNA group. The differences in operation time, intraoperative bleeding, immobilization duration, hospitalization time, Harris scores and postoperative complications including deep venous thrombosis, lung and urinary infection were analyzed. RESULTS: All patients were followed for 12.5-36.2 months [(28.0 ± 6.3) months)]. The operation time was (53.7 ± 15.2) min and (77.5 ± 16.8) min in PFNA group and CPH group, respectively (P < 0.05); intraoperative bleeding was (132.5 ± 33.2) mL and (286.3 ± 43.2) mL, respectively (P < 0.05); immobilization duration was (28.2 ± 3.7) days and (3.1 ± 1.2) days, respectively (P < 0.05); hospitalization time was (7.6 ± 1.8) days and (6.9 ± 2.2) days, respectively (P > 0.05); and the Harris scores after 1 year were (87.7 ± 7.9) points and (88.3 ± 9.2) points, respectively (P > 0.05). There was no significant difference in postoperative complications between the two groups (P > 0.05). CONCLUSION: Both PFNA and CPH are safe and effective treatments for femoral intertrochanteric fracture in elderly patients. Nonetheless, CPH allows faster mobilization and recovery. TRIAL REGISTRATION: Registration Number: ChiCTR1900022846 . Reg Date:2019-04-26 00:27:33 Retrospective registration.

A Generic Method for Preparing Hollow Mesoporous Silica Catalytic Nanoreactors with Metal Oxide Nanoparticles inside Their Cavities.

We report a facile and generic method for the synthesis of hollow mesoporous silica nanoreactors (HMSNs) with small-sized metal oxide nanoparticles (NPs) inside their cavities. They were made by deposition of silica onto metal-containing charge-driven polymer micelles and subsequent calcination. The micelles consist of 1) negatively charged supramolecular polyelectrolyte chains of bis-ligand-bound metal ions, and 2) water-soluble, neutral/positive diblock copolymers. Owing to the facile coordination between transition-metal ion and the employed bidentate ligand, a series of HMSNs with <2 nm Mx Oy NPs inside cavities (M=Mn, Co, Ni, Cu, or Zn) were obtained by simply varying the metal ions inside the micelles. The developed method circumvents the pre- and post-synthesis of metal oxide NPs; after calcination, hollow mesoporous nanostructures containing small-sized metal oxide NPs inside their cavities are directly obtained. The Cox Oy -functionalized HMSNs catalyze the degradation of various dyes with H2 O2 .

Developmental hip dysplasia treated by total hip arthroplasty using a cementless Wagner cone stem in young adult patients with a small physique.

BACKGROUND: Developmental hip dysplasia (DDH) may lead to severe acetabular and femoral abnormalities that can render total hip arthroplasty (THA) challenging, especially in DDH patients with a small physique. Most conventional cemented or cementless femoral components are often difficult to implant in the narrow femoral canal and require slight version correction during surgery. The aim of this study was to present the mid-term results of THA in the treatment of DDH patients with a small physique using a cementless Wagner cone prosthesis (Zimmer®, US). METHODS: Between January 2006 and March 2010, we retrospectively reviewed 50 patients who were treated at our center. A total of 50 patients (52 hips; 45 women, five men; mean age 32.5 years; range 27 to 38 years) who underwent THA were observed. The mean femoral medullary canal dimension at the isthmus was 7.6 mm (range 6.0 to 8.7). According to the Crowe classification, 19 hips presented dysplasia of grade I, while 33 presented dysplasia of grade II. All patients were treated with THA using a cementless Wagner cone prosthesis. Clinical and radiologic evaluations were performed on all patients. RESULTS: The mean duration of follow-up was 7.7 years (range 5.4 to 10.5). The Harris hip score (HHS) improved from 63 ± 9 (range 55 to 70) pre-operatively to 92 ± 8 (range 88 to 100) at the last follow-up. The HHS at the most recent follow-up was excellent in 66% of patients (34 hips), good in 26% (14 hips), fair in 6% (3 hips), and poor in 2% (1 hip). Radiographic evaluation demonstrated excellent osteointegration of the implants. Stem subsidence was present in three stems, and the range of stem subsidence was 2 mm in two stems (3.9%) and 3 mm in one stem (1.9%). Femoral osteolysis was observed in nine hips (18%) in the proximal zones, and no distal osteolysis was noted. Heterotopic ossification was observed in three hips (5.8%); of these, two were classified as Brooker's grade 1, and one was classified as Brooker's grade 2 at the most recent follow-up. None of the implants were revised. CONCLUSIONS: Based on the tapered shape and free setting of anteversion, the Wagner cone femoral stem facilitates its implantation in dysplastic hips. Therefore, this series of short stems with a smaller diameter can ensure safe implantation in narrow medullary canals, especially in young DDH patients with a small physique. TRIAL REGISTRATION: Registration Number: ChiCTR-ORC-17011181 . Reg Date: 2017-04-19 00:44:59 Retrospective registration.

The application of powerful promoters to enhance gene expression in industrial microorganisms.

Production of useful chemicals by industrial microorganisms has been attracting more and more attention. Microorganisms screened from their natural environment usually suffer from low productivity, low stress resistance, and accumulation of by-products. In order to overcome these disadvantages, rational engineering of microorganisms to achieve specific industrial goals has become routine. Rapid development of metabolic engineering and synthetic biology strategies provide novel methods to improve the performance of industrial microorganisms. Rational regulation of gene expression by specific promoters is essential to engineer industrial microorganisms for high-efficiency production of target chemicals. Identification, modification, and application of suitable promoters could provide powerful switches at the transcriptional level for fine-tuning of a single gene or a group of genes, which are essential for the reconstruction of pathways. In this review, the characteristics of promoters from eukaryotic, prokaryotic, and archaea microorganisms are briefly introduced. Identification of promoters based on both traditional biochemical and systems biology routes are summarized. Besides rational modification, de novo design of promoters to achieve gradient, dynamic, and logic gate regulation are also introduced. Furthermore, flexible application of static and dynamic promoters for the rational engineering of industrial microorganisms is highlighted. From the perspective of powerful promoters in industrial microorganisms, this review will provide an extensive description of how to regulate gene expression in industrial microorganisms to achieve more useful goals.

[Sirtuin type 1 signaling pathway mediates the effect of diosgenin on chondrocyte metabolisms in osteoarthritis].

OBJECTIVE: To observe the effect of sirtuin type 1 (SIRT1) signaling pathway on chondrocyte metabolism and mitochondrial oxidative stress, to explore roles of SIRT1 signaling pathway and diosgenin (Dgn) in pathogenesis of osteoarthritis (OA).
 Methods: To establish C57BL/6 mouse (13.5-18.0 g) model of OA. The chondrocytes were randomly assigned into 4 groups: an OA group, a Dgn+OA group, a Dgn+Sirtinol+OA group, and a Sirtinol+OA group. Expressions of SIRT1, acetylation-regulated transcription factor 1 (Ac-FOXO1), and Bax were detected in the 4 groups by Western blot. Changes in levels of succinate dehydrogenase, cytochrome C oxidase, and superoxide dismutase of mitochondrion in the 4 groups were observed.
 Results: Compared with the OA group, SIRT1 protein expression was increased in the OA+Dgn group (P<0.05), while the Ac-FOXO1 and Bax protein expressions were decreased (P<0.05), the SDH and COX expressions were decreased, and the SOD content was decreased (P<0.05). Compared with the OA+Dgn group, the SIRT1 expression in the OA+Dgn+Sirtinol group and the Sirtinol+OA group was increased (P<0.05), while the Ac-FOXO1 and Bax protein expressions were decreased (P<0.05). The SDH and COX expressions were decreased, the SOD content was decreased, the SDH and COX expressions were increased, and the SOD content was increased in the OA group (All P<0.05), while those indexes were reversed in the OA+Dgn+Sirtinol group and the Sirtinol+OA group (All P<0.05).
 Conclusion: The SIRT1 signaling pathway and OA are closely related to changes in chondrocyte apoptosis, in which Dgn may play a protective role in anti-OA by activating SIRT1 signaling pathway to inhibit apoptosis of chondrocytes and to increase chondrocyte mitochondrial oxidative stress capacity.

Characterization of mutants of a tyrosine ammonia-lyase from Rhodotorula glutinis.

In the phenylpropanoid production process, p-coumaric acid is the most important intermediate metabolite. It is generally accepted that the activity of tyrosine ammonia-lyase (TAL), which converts L-tyrosine to p-coumaric acid, represents the rate-limiting step. Therefore, an error-prone PCR-based random mutagenesis strategy was utilized for screening variants with higher catalytic activity. After rounds of screening, three variant enzymes were obtained, exhibiting improved production rates of 41.2, 37.1, and 38.0 %, respectively. Variants associated with increased expression level (S9N), improved catalytic efficiency (A11T), and enhanced affinity between TAL and L-tyrosine (E518V) were identified as beneficial amino acid substitutions by site-directed mutagenesis. Combining all of the beneficial amino acid substitutions, a variant, MT-S9N/-A11T/-E518V, exhibiting the highest catalytic activity was obtained. The K m value of MT-S9N/-A11T/-E518V decreased by 25.4 % compare to that of wild-type, while the activity, k cat/K m, and p-coumaric-acid yield were improved by 36.5, 31.2, and 65.9 %, respectively. Furthermore, the secondary structure of the 5'-end of MT-S9N mRNA and the three-dimensional protein structure of MT-E518V were modeled. Therefore, two potential mechanisms were speculated: (1) a simplified mRNA 5'-end secondary structure promotes TAL expression and (2) anchoring the flexible loop region (Glu325-Arg336) to maintain the active-site pocket opening ensures easy access by the L-tyrosine to the active site and thus improves p-coumaric acid yields.