Correlation Analysis of Estrogen Levels and Psychological Status Among Patients with Uterine Fibroids.

Objective: This study aims to investigate the correlation between estrogen levels and psychological distress, focusing on depression and anxiety symptoms among patients diagnosed with uterine fibroids. Methods: The study employed a retrospective design and enrolled a cohort comprising 50 patients diagnosed with uterine fibroids and 50 healthy individuals as controls. Serum estradiol levels were quantified using a chemiluminescent immunoassay technique one month before surgery in the patient group. Depression and anxiety levels were evaluated using the Self-Rating Depression Scale (SDS) and the Self-Rating Anxiety Scale (SAS), respectively. Results: Significant differences in SDS scores, SAS scores, and serum estradiol levels emerged between the patient and control groups (P < .05). Patients exhibited higher SDS and SAS scores alongside elevated serum estradiol levels. Correlation analysis unveiled a negative association between SAS scores and estrogen levels among patients (r = -0.724, P = .013), suggesting a rise in anxiety levels with declining estrogen levels. Similarly, a negative correlation surfaced between SDS scores and estrogen levels among patients (r = -0.624, P = .016), indicating increased depressive symptoms as estrogen levels decrease. Conversely, no noteworthy correlations were demonstrated between anxiety or depressive symptoms and estrogen levels in the control group. Conclusion: Reduced estrogen levels were linked to heightened anxiety and depressive symptoms in patients with uterine fibroids. These findings suggest a plausible connection between estrogen hormone levels and psychological well-being, particularly concerning anxiety and depression. Further exploration of this association is warranted to shed light on potential therapeutic interventions targeting hormonal regulation to improve psychological distress in affected individuals.

Mechanically Robust and Flame-Retardant Superhydrophobic Textiles with Anti-Biofouling Performance.

The attachment of bio-fluids to surfaces promotes the transmission of diseases. Superhydrophobic textiles may offer significant advantages for reducing the adhesion of bio-fluids. However, they have not yet found widespread use because dried remnants adhere strongly and have poor mechanical or chemical robustness. In addition, with the massive use of polymer textiles, features such as fire and heat resistance can reduce the injuries and losses suffered by people in a fire accident. We developed a superhydrophobic textile covered with a hybrid coating of titanium dioxide and polydimethylsiloxane (TiO2/PDMS). Such a textile exhibits low adhesion to not only bio-fluids but also dry blood. Compared to a hydrophilic textile, the peeling force of the coated textile on dried blood is 20 times lower. The textile's superhydrophobicity survives severe treatment by sandpaper (400 mesh) at high pressure (8 kPa) even if some of its microstructures break. Furthermore, the textile shows excellent heat resistance (350 °C) and flame-retardant properties as compared to those of the untreated textile. These benefits can greatly inhibit the flame spread and reduce severe burns caused by polymer textiles adhering to the skin when melted at high temperatures.

Impact of target-mediated drug disposition on hetrombopag pharmacokinetics and pharmacodynamics in Chinese healthy subjects and patients with chronic idiopathic thrombocytopenic purpura.

AIMS: The pharmacokinetics (PK) of hetrombopag were found to be nonlinear across evaluated dose ranges. The aim of this study was to develop a mechanism-based population pharmacokinetic/pharmacodynamic (PopPK/PD) model and to provide a reasonable expected therapeutic dose for a future confirmatory clinical study of hetrombopag. METHODS: Nonlinear mixed-effects modelling was performed using pooled 2168 hetrombopag concentrations and 1526 platelet counts from 72 healthy subjects and 32 chronic idiopathic thrombocytopenic purpura (ITP) patients from two phase I studies and one phase II study. The final model was evaluated via goodness-of-fit plots, visual predictive check and nonparametric bootstrap. Simulations from the validated PopPK/PD model were used to devise an expected therapeutic dose for later confirmatory clinical study. RESULTS: The pharmacokinetic data of hetrombopag were well described by a modified target-mediated drug disposition (TMDD) model with dual sequential first-order absorption. Mean parameter estimates (interindividual variability) were CL/F 7.66 L/h (63.5%), Vc /F 30.0 L (77.2%) and Kdeg 0.693/h (87.1%). The pharmacodynamic profile was well described by a five-compartment lifespan model with four-transit and one-platelet compartments. Simulation results suggested that chronic ITP patients following 10 mg once-daily hetrombopag would able to achieve an ideal platelet count level (50-200 × 109 /L). CONCLUSION: TMDD was the primary reason leading to nonlinear PK profile of hetrombopag. Our PK/PD modelling and simulation results support 10 mg once-daily as the recommended therapeutic dose for chronic ITP patients in subsequent confirmatory clinical study of hetrombopag.

Multi-omics reveals a relationship between endometrial amino acid metabolism and autophagy in women with recurrent miscarriage†.

Deterioration of the endometrial environment is an essential cause of recurrent miscarriage (RM). However, current studies in terms of endometrial amino acid metabolic characterization and autophagy are still inadequate. We tried to (1) identify the alternation in metabolite profiles in the RM endometrium; (2) investigate the expression of autophagy-related proteins in RM; and (3) elucidate the association between amino acid metabolism and autophagy in RM. Our results showed that glutamine metabolites were up-regulated in the endometrium of RM women. The levels of autophagy-associated proteins, LC3B, ATG12, and Beclin-1, were significantly higher in RM. Hemostasis, autophagy and IFNα signaling were the top three differentially activated signaling pathways between women with RM and normal pregnancy. Interestingly the expression of AMPK and GCN2 was significantly up-regulated in the endometrium of women with RM, and the same expression trend was also observed in the human endometrial stromal cells cultured in glutamine deprivation medium. Furthermore, inhibition of AMPK decreased the level of GCN2, indicating a positive correlation between GCN2 and AMPK. The expression of GCN2 was consistent with the expression of ATG12 and beclin-1; however, it was opposite to that of p62. Exposure to glutamine deprivation increased the level of LC3B, GCN2, ATG12, and beclin-1. Altogether, these findings suggested significant crosstalk between amino acid metabolism and autophagy. In summary, our data suggested that aberrant crosstalk between amino acid metabolism and autophagy may contribute to the impaired endometrial microenvironment of RM. Our study may provide new insight into the diagnosis of RM due to endometrial factors.

A visualized clinical model predicting good quality blastocyst development in the first IVF/ICSI cycle.

RESEARCH QUESTION: Is it possible to establish a visualized clinical model predicting good quality blastocyst (GQB) formation for patients in their first IVF/intracytoplasmic sperm injection (ICSI) cycle? DESIGN: A total of 4783 patients in their first IVF/ICSI cycle between January 2015 and December 2019 were retrospectively included and randomly divided into the training set (n = 3826) and the testing set (n = 957) in an 8:2 ratio. The least absolute shrinkage and selection operator (LASSO) regression was adopted to select the most critical predictors for GQB formation to construct a visualized nomogram model based on the data of patients in the training set. Receiver operating characteristic and calibration curves were used to evaluate the predictive accuracy and discriminative ability. The performance of the model was also validated on independent data from patients treated in the testing set. RESULTS: Maternal age, maternal serum anti-Müllerian hormone (MsAMH) concentration and the number of oocytes retrieved were highlighted as critical predictors of GQB development and were incorporated into the nomogram model. Based on the area under the curve (AUC) values, the predictive ability for ≥1, ≥3 and ≥5 GQB were 0.831, 0.734 and 0.748, respectively. The calibration curve also showed high concordance between the observed and predicted results. The AUC for predicting ≥1, ≥3 and ≥5 GQB in the testing set were 0.805, 0.695 and 0.707, respectively, which were similar to those for the training set. CONCLUSIONS: The visualized nomogram model provides great predictive value for GQB development in patients in their first IVF/ICSI cycle and can be used to improve clinical counselling.

Manipulating the position of DNA expression cassettes using location tags fused to dCas9 (Cas9-Lag) to improve metabolic pathway efficiency.

BACKGROUND: Deactivated Cas9 (dCas9) led to significant improvement of CRISPR/Cas9-based techniques because it can be fused with a variety of functional groups to form diverse molecular devices, which can manipulate or modify target DNA cassettes. One important metabolic engineering strategy is to localize the enzymes in proximity of their substrates for improved catalytic efficiency. In this work, we developed a novel molecular device to manipulate the cellular location of specific DNA cassettes either on plasmids or on the chromosome, by fusing location tags to dCas9 (Cas9-Lag), and applied the technique for synthetic biology applications. Carotenoids like ß-carotene serve as common intermediates for the synthesis of derivative compounds, which are hydrophobic and usually accumulate in the membrane compartment. RESULTS: Carotenoids like ß-carotene serve as common intermediates for the synthesis of derivative compounds, which are hydrophobic and usually accumulate in the membrane components. To improve the functional expression of membrane-bound enzymes and localize them in proximity to the substrates, Cas9-Lag was used to pull plasmids or chromosomal DNA expressing carotenoid enzymes onto the cell membrane. For this purpose, dCas9 was fused to the E. coli membrane docking tag GlpF, and gRNA was designed to direct this fusion protein to the DNA expression cassettes. With Cas9-Lag, the zeaxanthin and astaxanthin titer increased by 29.0% and 26.7% respectively. Due to experimental limitations, the electron microscopy images of cells expressing Cas9-Lag vaguely indicated that GlpF-Cas9 might have pulled the target DNA cassettes in close proximity to membrane. Similarly, protein mass spectrometry analysis of membrane proteins suggested an increased expression of carotenoid-converting enzymes in the membrane components. CONCLUSION: This work therefore provides a novel molecular device, Cas9-Lag, which was proved to increase zeaxanthin and astaxanthin production and might be used to manipulate DNA cassette location.

A programmable CRISPR/Cas9-based phage defense system for Escherichia coli BL21(DE3).

Escherichia coli BL21 is arguably the most popular host for industrial production of proteins, and industrial fermentations are often plagued by phage infections. The CRISPR/Cas system is guided by a gRNA to cleave a specific DNA cassette, which can be developed into a highly efficient programable phage defense system. In this work, we constructed a CRISPR/Cas system targeting multiple positions on the genome of T7 phage and found that the system increased the BL21's defense ability against phage infection. Furthermore, the targeted loci on phage genome played a critical role. For better control of expression of CRISPR/Cas9, various modes were tested, and the OD of the optimized strain BL21(pT7cas9, pT7-3gRNA, prfp) after 4 h of phage infection was significantly improved, reaching 2.0, which was similar to the control culture without phage infection. Although at later time points, the defensive ability of CRISPR/Cas9 systems were not as obvious as that at early time points. The viable cell count of the engineered strain in the presence of phage was only one order of magnitude lower than that of the strain with no infection, which further demonstrated the effectiveness of the CRISPR/Cas9 phage defense system. Finally, the engineered BL21 strain under phage attack expressed RFP protein at about 60% of the un-infected control, which was significantly higher than the parent BL21. In this work, we successfully constructed a programable CRISPR/Cas9 system to increase the ability of E. coli BL21's to defend against phage infection, and created a resistant protein expression host. This work provides a simple and feasible strategy for protecting industrial E. coli strains against phage infection.

miR-146a promotes proliferation, invasion, and epithelial-to-mesenchymal transition in oral squamous carcinoma cells.

Epithelial-to-mesenchymal transition (EMT) is key to invasion and metastasis by oral squamous carcinoma (OSCC) cells. MicroRNAs (miRNAs) such as miRNA-146a are known to be upregulated in OSCC. However, it is unclear whether they are involved in driving EMT. Here, we investigated the effect of miR-146a overexpression on proliferation, migration, and EMT in OSCC cells. OSCC cells were transfected with a plasmid expressing miR-146a precursor. Cell lines that stably overexpressed miRNA-146a were assessed for proliferation, colony formation, and invasiveness in vitro. Expression of markers and regulators of EMT, cell motility, and invasion were measured by qRT-PCR and western blot. Potential miRNA-146a binding sites in the 3'UTR of ST8SIA4 were identified by bioinformatic analysis. To confirm that miRNA-146a binds to and regulates ST8SIA4, we transfected OSCC cell lines with miRNA-146a mimics and a luciferase reporter construct containing either the wild type or mutant 3'UTR of ST8SIA4. OSCC cell lines that overexpressed miR-146a displayed higher proliferation, colony formation, invasion, and MMP-2 activity than cells transfected with a control vector. Overexpression of miR-146a also decreased expression of the epithelial cell marker E-cadherin and increased expression of Twist1, a transcription factor that promotes EMT, as well as markers associated with mesenchymal cells (vimentin and N-cadherin) and tumor invasion (p-paxillin and p-cortactin). Luciferase expression was lower in OSCC cells transfected with miRNA-146a mimics or with luciferase constructs carrying the wild type, but not mutant, 3'UTR of ST8SIA4. Overexpression of miR-146a promotes EMT phenotypes and may drive tumorigenesis and progression in OSCC, making it a useful target for future OSCC treatments.

Control of Droplet Evaporation on Oil-Coated Surfaces for the Synthesis of Asymmetric Supraparticles.

Controlling the droplet evaporation on surfaces is desired to get uniform depositions of materials in many applications, for example, in two- and three-dimensional printing and biosensors. To explore a new route to control droplet evaporation on surfaces and produce asymmetric particles, sessile droplets of aqueous dispersions were allowed to evaporate from surfaces coated with oil films. Here, we applied 1-50 µm thick films of different silicone oils. Two contact lines were observed during droplet evaporation: an apparent liquid-liquid-air contact line and liquid-liquid-solid contact line. Because of the oil meniscus covering part of the rim of the drop, evaporation at the periphery is suppressed. Consequently, the droplet evaporates mainly in the central region of the liquid-air interface rather than at the droplet's edge. Colloidal particles migrate with the generated upward flow inside the droplet and are captured by the receding liquid-air interface. A uniform deposition ultimately forms on the substrate. With this straightforward approach, asymmetric supraparticles have been successfully fabricated independent of particle species.

Construction of Escherichia coli cell factories for crocin biosynthesis.

BACKGROUND: Crocin is a carotenoid-derived natural product found in the stigma of Crocus spp., which has great potential in medicine, food and cosmetics. In recent years, microbial production of crocin has drawn increasing attention, but there were no reports of successful implementation. Escherichia coli has been engineered to produce various carotenoids, including lycopene, ß-carotene and astaxanthin. Therefore, we intended to construct E. coli cell factories for crocin biosynthesis. RESULTS: In this study, a heterologous crocetin and crocin synthesis pathway was first constructed in E. coli. Firstly, the three different zeaxanthin-cleaving dioxygenases CsZCD, CsCCD2 from Crocus sativus, and CaCCD2 from Crocus ancyrensis, as well as the glycosyltransferases UGT94E5 and UGT75L6 from Gardenia jasminoides, were introduced into zeaxanthin-producing E. coli cells. The results showed that CsCCD2 catalyzed the synthesis of crocetin dialdehyde. Next, the aldehyde dehydrogenases ALD3, ALD6 and ALD9 from Crocus sativus and ALD8 from Neurospora crassa were tested for crocetin dialdehyde oxidation, and we were able to produce 4.42 mg/L crocetin using strain YL4(pCsCCD2-UGT94E5-UGT75L6,pTrc-ALD8). Glycosyltransferases from diverse sources were screened by in vitro enzyme activity assays. The results showed that crocin and its various derivatives could be obtained using the glycosyltransferases YjiC, YdhE and YojK from Bacillus subtilis, and the corresponding genes were introduced into the previously constructed crocetin-producing strain. Finally, crocin-5 was detected among the fermentation products of strain YL4(pCsCCD2-UGT94E5-UGT75L6,pTrc-ALD8,pET28a-YjiC-YdhE-YojK) using HPLC and LC-ESI-MS. CONCLUSIONS: A heterologous crocin synthesis pathway was constructed in vitro, using glycosyltransferases from the Bacillus subtilis instead of the original plant glycosyltransferases, and a crocetin and crocin-5 producing E. coli cell factory was obtained. This research provides a foundation for the large-scale production of crocetin and crocin in E. coli cell factories.

Evaluation of two aneuploidy screening tests for chorionic villus samples: Multiplex ligation-dependent probe amplification and fluorescence in situ hybridization.

The vast majority of first-trimester pregnancy losses are the consequence of numerical aberrations in fetal chromosomes, which may involve nearly all chromosomes. Although commercial probes for all chromosomes are available for multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH) analyses, their use has rarely been reported for screening all 24 chromosomes for early fetal demise, especially by FISH. Here, we validated the ability of MLPA and FISH techniques as two low-cost aneuploidy screening methods for 24 chromosomes in 165 chorionic villus samples (CVSs). The results obtained by two methods were compared by the Chi-square test and the Kappa agreement test. Both methods gave conclusive results for all CVSs tested and showed highly consistent results (kappa = 0.890, p < 0.001). There was no statistically significant difference between the aneuploidy rate of the CVSs tested by the two methods (p = 0.180). Most of the samples showed fully concordant molecular karyotyping results (81.21%) between the two analytical methods, 10.91% had incompletely concordant results, and 7.88% had discordant results. The inconsistencies included segmental abnormalities, mosaicism, and polyploidy. Both assays used to screen 24 chromosomes were powerful techniques for detecting aneuploidy in CVSs. In terms of cost-effectiveness and diagnostic accuracy, the combination of subtelomeric (P036, P070) and centromeric (P181) MLPA assays is the better analytic strategy and follow-up analysis by FISH is recommended for MLPA-negative samples.

Responsive Ionogel Surface with Renewable Antibiofouling Properties.

The synthesis of ionogels with a responsive, self-replenishing surface for combating biofouling is described. Ionogels are prepared by infiltrating poly(vinylidene fluoride-co-hexafluoropropylene) with binary mixtures of ionic liquids (IL): 1-octadecyl-3-methylimidazolium bis(trifluoromethyl sulfonyl)imide ([C18 C1 im][NTf2 ], melting point Tm = 55 °C) and 1-hexyl-3-methylimidazolium bis(trifluoromethyl sulfonyl)imide ([C6 C1 im][NTf2 ], Tm = -9 °C). The IL mixtures release spontaneously from the gel matrix and eventually crystallize on the surface. This leads to self-replenishment of the surface of ionogels even after mechanical damage. The incorporation of [C6 C1 im][NTf2 ] provides the antimicrobial efficacy of ionogels while the crystals of [C18 C1 im][NTf2 ] serve as a skeleton maintaining [C6 C1 im][NTf2 ] on the surface. By heating, the ionogel surface transforms from solid to liquid-infused state-the removal of biofilms/bacteria developed under a long time of colonization is facilitated. The antimicrobial efficacy is maintained even after several cycles of biofilm formation and detachment. This work provides an opportunity to apply ionogels as functional coatings with renewable antibiofouling properties.

CARD9 downregulation suppresses the growth of oral squamous cell carcinoma by regulating NF-κB.

OBJECTIVE: To discover the expression pattern and potential underlying mechanism of the caspase recruitment domain-containing protein 9 (CARD9) in oral squamous cell carcinoma (OSCC). METHODS: Caspase recruitment domain-containing protein 9 expression was detected by qRT-PCR and Western blot in OSCC tissues and cells, and OSCC (CGHNC9 and OECM-1) cell lines were divided into control, NC siRNA, and CARD9 siRNA groups. Then, MTT, flow cytometry, wound-healing, and Transwell assays were carried out to determine the changes in cellular biological characteristics. Immunoblot assay was performed for the expressions of NF-κB pathway. Finally, we constructed the xenograft models in nude mice to validate the in vivo effect of CARD9 siRNA on OSCC cell growth. RESULTS: Caspase recruitment domain-containing protein 9 was upregulated in both OSCC tissues and cells, exhibiting a close relation with major clinicopathological features of OSCC patients. Transfection of CARD9 siRNA inhibited the proliferation, migration, and invasion of OSCC cells with the enhanced cell apoptosis, and meanwhile, CARD9, p-p65/p65, p-IKKα/IKKα, and p-IkBα/IkBα were downregulated. The tumor formation assay on nude mice also suggested that CARD9 siRNA might block the in vivo growth of OSCC cells. CONCLUSION: Caspase recruitment domain-containing protein 9 suppression results in the upregulation of NF-κB pathway with suppressed proliferation, migration, and invasion of OSCC cells and facilitates the apoptosis.

Prevalent genotypes of methylenetetrahydrofolate reductase (MTHFR) in recurrent miscarriage and recurrent implantation failure.

OBJECTIVE: To evaluate the association of two common methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms with recurrent miscarriage (RM) and repeated implantation failure (RIF) METHODS: The study comprised of 521 patients, with a history of RM (n = 370) or RIF (n = 151). One hundred forty-four women with fallopian tube blockages who had successfully conceived after the first in vitro fertilization embryo transfer treatment served as the control group. The MTHFR alleles, genotypes, and haplotypes were assessed in different groups. RESULTS: There was no difference in allele frequency and distribution of MTHFR polymorphisms between case and control patients. The 1298AA genotype was represented in a higher frequency, and 1298AC genotype was significantly lower in subfertile group when compared to the control group. A significant relationship was found between the 1298AC genotype and the RIF subgroup. The haplotype 677CC/1298AA was overrepresented in the RM subgroup (> 2 times) and haplotype 677CC/1298AC was underrepresented in the RIF subgroup (P < 0.05). Nevertheless, these two haplotypes were not connected to fertilization and embryo cleavage rates. CONCLUSION: Our findings indicate that the MTHFR gene polymorphism might play a role in the etiology of patients with RM or RIF. No adverse effects of different MTHFR haplotypes on embryo development were detected. Further studies on the biological role are needed to better understand the susceptibility to pregnancy complications.

Membrane engineering - A novel strategy to enhance the production and accumulation of ß-carotene in Escherichia coli.

Carotenoids are a class of terpenes of commercial interest that exert important biological functions. While various strategies have been applied to engineer ß-carotene production in microbial cell factories, no work has been done to study and improve the storage of hydrophobic terpene products inside the heterologous host cells. Although the membrane is thought to be the cell compartment that accumulates hydrophobic terpenes such as ß-carotene, direct evidence is still lacking. In this work, we engineered the membrane of Escherichia coli in both its morphological and biosynthetic aspects, as a means to study and improve its storage capacity for ß-carotene. Engineering the membrane morphology by overexpressing membrane-bending proteins resulted in a 28% increase of ß-carotene specific producton value, while engineering the membrane synthesis pathway led to a 43% increase. Moreover, the combination of these two strategies had a synergistic effect, which caused a 2.9-fold increase of ß-carotene specific production value (from 6.7 to 19.6mg/g DCW). Inward membrane stacks were observed in electron microscopy images of the engineered E. coli cells, which indicated that morphological changes were associated with the increased ß-carotene storage capacity. Finally, membrane separation and analysis confirmed that the increased ß-carotene was mainly accumulated within the cell membrane. This membrane engineering strategy was also applied to the ß-carotene hyperproducing strain CAR025, which led to a 39% increase of the already high ß-carotene specific production value (from 31.8 to 44.2mg/g DCW in shake flasks), resulting in one of the highest reported specific production values under comparable culture conditions. The membrane engineering strategy developed in this work opens up a new direction for engineering and improving microbial terpene producers. It is quite possible that a wide range of strains used to produce hydrophobic compounds can be further improved using this novel engineering strategy.

Fabrication of Superhydrophobic Surfaces with Controllable Electrical Conductivity and Water Adhesion.

A facile and versatile strategy for fabricating superhydrophobic surfaces with controllable electrical conductivity and water adhesion is reported. "Vine-on-fence"-structured and cerebral cortex-like superhydrophobic surfaces are constructed by filtering a suspension of multiwalled carbon nanotubes (MWCNTs), using polyoxymethylene nonwovens as the filter paper. The nonwovens with micro- and nanoporous two-tier structures act as the skeleton, introducing a microscale structure. The MWCNTs act as nanoscale structures, creating hierarchical surface roughness. The surface topography and the electrical conductivity of the superhydrophobic surfaces are controlled by varying the MWCNT loading. The vine-on-fence-structured surfaces exhibit "sticky" superhydrophobicity with high water adhesion. The cerebral cortex-like surfaces exhibit self-cleaning properties with low water adhesion. The as-prepared superhydrophobic surfaces are chemically resistant to acidic and alkaline environments of pH 2-12. They therefore have potential in applications such as droplet-based microreactors and thin-film microextraction. These findings aid our understanding of the role that surface topography plays in the design and fabrication of superhydrophobic surfaces with different water-adhesion properties.

Type IIs restriction based combinatory modulation technique for metabolic pathway optimization.

BACKGROUND: One of the most important research subjects of metabolic engineering is pursuing a balanced metabolic pathway, which is the basis of an efficient cell factory. In this work, we dedicated to develop a simple and efficient technique to modulate expression of multiple genes simultaneously, and select for the optimal regulation pattern. RESULTS: A Type IIs restriction based combinatory modulation (TRCM) technique was designed and established in the research. With this technique, a plasmid library containing variably regulated mvaE, mvaS, mvaK 1 , mvaD and mvaK 2 of the mevalonate (MVA) pathway were obtained and transformed into E. coli DXS37-IDI46 to obtain a ß-carotene producer library. The ratio of successfully assembled plasmids was determined to be 35%, which was increased to 100% when color based pre-screening was applied. Representative strains were sequenced to contain diverse RBSs as designed to regulate expression of MVA pathway genes. A relatively balanced MVA pathway was achieved in E. coli cell factory to increase the ß-carotene yield by two fold. Furthermore, the approximate regulation pattern of this optimal MVA pathway was illustrated. CONCLUSIONS: A TRCM technique for metabolic pathway optimization was designed and established in this research, which can be applied to various applications in terms of metabolic pathway regulation and optimization.

Comparative study in infertile couples with and without Chlamydia trachomatis genital infection.

BACKGROUND: Chlamydia, caused by the bacterium Chlamydia trachomatis(C. trachomatis), is the most common sexually transmitted disease. The incidence is not clear due to the asymptomatic nature of early stage of infections. The incidence of Chlamydia has not been fully investigated in the Chinese Han population. Since chronic infection with can C. trachomatis can lead to infertility in males and females, it is important to determine the impact of infection on clinical outcomes. The aim of this study is to explore the epidemiology of C. trachomatis in subfertile couples and to determine whether infections will adversely affect clinical outcomes after assisted reproduction technique (ART) treatment. METHODS: Subfertile patients (n = 30760) were screened in the research for C. trachomatis in our center from January 2010 to December 2014. C. trachomatis-specific DNA was detected by Taq-man PCR from semen or swabs from the urethral, endocervix or vaginal. The control group consisted of 1140 subfertile patients without C. trachomatis infection. The prevalence and characteristics of C. trachomatis were identified for subfertile couples and clinical outcomes were collected and analyzed. A retrospective study was performed. RESULTS: Nine hundred and seventy patients were diagnosed with C. trachomatis infection, and the overall prevalence was 3.15% in the most recent five years, with a yearly increasing. The incidence was a higher in the second half of the year (3.40%) compared to the first half (2.69%). The age group with the highest-risk of infection with C. trachomatis was between 26 to 35 years old, and in about one third of the couples, both partners were infected. The basic parameters and clinical outcomes were not statistically significant between different the groups (P > 0.05), even though some minor data were different (P < 0.05). CONCLUSIONS: C. trachomatis is a common infection in subfertile people and it is essential to test for this organism in ART couples' screening. This study identified no adverse on clinical outcomes after successful treatment of C. trachomatis infection, regardless of gender, age and number of C. trachomatis copies.

Combinatory optimization of chromosomal integrated mevalonate pathway for ß-carotene production in Escherichia coli.

BACKGROUND: Plasmid expression is a popular method in studies of MVA pathway for isoprenoid production in Escherichia coli. However, heterologous gene expression with plasmid is often not stable and might burden growth of host cells, decreases cell mass and product yield. In this study, MVA pathway was divided into three modules, and two heterologous modules were integrated into the E. coli chromosome. These modules were individually modulated with regulatory parts to optimize efficiency of the pathway in terms of downstream isoprenoid production. RESULTS: MVA pathway modules Hmg1-erg12 operon and mvaS-mvaA-mavD1 operon were integrated into E. coli chromosome followed by modulation with promoters with varied strength. Along with activation of atoB, a 26% increase of ß-carotene production with no effect on cell growth was obtained. With a combinatory modulation of two key enzymes mvas and Hmg1 with degenerate RBS library, ß-carotene showed a further increase of 51%. CONCLUSIONS: Our study provides a novel strategy for improving production of a target compound through integration and modulation of heterologous pathways in both transcription and translation level. In addition, a genetically hard-coded chassis with both efficient MEP and MVA pathways for isoprenoid precursor supply was constructed in this work.

Development of a fast and easy method for Escherichia coli genome editing with CRISPR/Cas9.

BACKGROUND: Microbial genome editing is a powerful tool to modify chromosome in way of deletion, insertion or replacement, which is one of the most important techniques in metabolic engineering research. The emergence of CRISPR/Cas9 technique inspires various genomic editing methods. RESULTS: In this research, the goal of development of a fast and easy method for Escherichia coli genome editing with high efficiency is pursued. For this purpose, we designed modular plasmid assembly strategy, compared effects of different length of homologous arms for recombination, and tested different sets of recombinases. The final technique we developed only requires one plasmid construction and one transformation of practice to edit a genomic locus with 3 days and minimal lab work. In addition, the single temperature sensitive plasmid is easy to eliminate for another round of editing. Especially, process of the modularized editing plasmid construction only takes 4 h. CONCLUSION: In this study, we developed a fast and easy genome editing procedure based on CRISPR/Cas9 system that only required the work of one plasmid construction and one transformation, which allowed modification of a chromosome locus within 3 days and could be performed continuously for multiple loci.