Flexible Seven-in-One Microsensor Embedded in High-Pressure Proton Exchange Membrane Water Electrolyzer for Real-Time Microscopic Monitoring.

The voltage, current, temperature, humidity, pressure, flow, and hydrogen in the high-pressure proton exchange membrane water electrolyzer (PEMWE) can influence its performance and life. For example, if the temperature is too low to reach the working temperature of the membrane electrode assembly (MEA), the performance of the high-pressure PEMWE cannot be enhanced. However, if the temperature is too high, the MEA may be damaged. In this study, the micro-electro-mechanical systems (MEMS) technology was used to innovate and develop a high-pressure-resistant flexible seven-in-one (voltage, current, temperature, humidity, pressure, flow, and hydrogen) microsensor. It was embedded in the upstream, midstream, and downstream of the anode and cathode of the high-pressure PEMWE and the MEA for the real-time microscopic monitoring of internal data. The aging or damage of the high-pressure PEMWE was observed through the changes in the voltage, current, humidity, and flow data. The over-etching phenomenon was likely to occur when this research team used wet etching to make microsensors. The back-end circuit integration was unlikely to be normalized. Therefore, this study used lift-off process to further stabilize the quality of the microsensor. In addition, the PEMWE is more prone to aging and damage under high pressure, so its material selection is very important.

Development and validation of a Systemic Sclerosis Health Literacy Scale.

Background and aim: Health literacy levels are strongly associated with clinical outcomes and quality of life in patients with chronic diseases, and patients with limited health literacy often require more medical care and achieve poorer clinical outcomes. Among the large number of studies on health literacy, few studies have focused on the health literacy of people with systemic sclerosis (SSc), and there is no specific tool to measure health literacy in this group. Therefore, this study plans to develop a health literacy scale for patients with SSc. Methods: This study included 428 SSc patients from the outpatient and inpatient departments of the Department of Rheumatology and Immunology, the first affiliated Hospital of Anhui Medical University and the first affiliated Hospital of University of Science and Technology of China. The formulation of the scale was completed by forming the concept of health literacy of SSc patients, establishing the item pool, screening items, and evaluating reliability and validity. Classical measurement theory was used to screen items, factor analysis was used to explore the construct validity of the scale, and Cronbach's alpha coefficient was used to assess the internal consistency. Results: Our study population was predominantly middle-aged women, with a male to female ratio of 1:5.7 and a mean age of 51.57 ± 10.99. A SSc Health Literacy scale with 6 dimensions and 30 items was developed. The six dimensions are clinic ability, judgment/evaluation information ability, access to information ability, social support, treatment compliance and application information ability. The Cronbach's alpha coefficient of the scale is 0.960, retest reliability is 0.898, split-half reliability is 0.953, content validity is 0.983, which has good reliability and validity. Conclusion: The Systemic Sclerosis Health Literacy Scale may become a valid tool to evaluate the health literacy level of patients with SSc.

High-Pressure-Resistant Flexible Seven-in-One Microsensor Embedded in High-Pressure Proton Exchange Membrane Water Electrolyzer for Real-Time Microscopic Measurement.

The high-pressure proton exchange membrane water electrolyzer (PEMWE) used for hydrogen production requires a high-operating voltage, which easily accelerates the decomposition of hydrogen molecules, resulting in the aging or failure of the high-pressure PEMWE. As the high-pressure PEMWE ages internally, uneven flow distribution can lead to large temperature differences, reduced current density, flow plate corrosion, and carbon paper cracking. In this study, a new type of micro hydrogen sensor is developed with integrated flexible seven-in-one (voltage; current; temperature; humidity; flow; pressure; and hydrogen) microsensors.

Isolation of an ectoine-producing Sinobaca sp. and identification of genes that are involved in ectoine biosynthesis.

A moderate halophilic bacterium that could accumulate ectoine and hydroxyectoine was isolated from soil near a salt mine and was identified as a Sinobaca sp. (designed strain H24) according to 16S rRNA gene sequence analysis. The bacterium grew well in the presence of 1-2 M NaCl, while growth in a medium that contained 2 M NaCl led to higher accumulation of ectoines. The yields of ectoine and hydroxyectoine by Sinobaca sp. H24 reached 11.27 mg/l and 1.34 mg/l, respectively, when cultured in the following medium: NaCl (2 M), peptone (5 g/l), yeast extract (1 g/l), NH4Cl (0.02 M), KH2PO4 (1 M), K2HPO4 (0.1 M), and glycerol (1% w/v). Genes that are involved in ectoine biosynthesis of Sinobaca sp. H24 were also identified, and their sequences were determined by a metagenomics approach. The results demonstrated that Sinobaca sp. H24 possesses ectoine metabolism genes for both ectoine biosynthesis (ectA, ectB, ectC, and ectD) and ectoine degradation (doeA). Genes that are related to ectoine biosynthesis, such as lysC and asd, were also characterized. The identification and characterization results for ectoine/hydroxyectoine biosynthesis genes are in agreement with the physiology of Sinobaca sp. H24 as a potential candidate for ectoine production for industrial applications. This report established for the first time the accumulation of ectoine/hydroxyectoine in Sinobaca sp. and characterized the genes that are involved in ectoine/hydroxyectoine biosynthesis in Sinobaca sp. H24.

A Method to Realize Efficient Deep-Red Phosphorescent OLEDs with a Broad Spectral Profile and Low Operating Voltages.

Organic light-emitting diodes (OLEDs) used as phototherapy light sources require sufficient spectral distribution in the effective wavelength ranges and low operating voltages. Herein, a double emitting layer structure consisting of a red-emitting Ir(piq)2acac and a deep-red Ir(fliq)2acac was designed to generate a broad electroluminescence spectrum. An efficient TCTA:CN-T2T exciplex system was used as the host of the emitting layer, facilitating effective energy transfer from the exciplex host to the red and deep-red phosphors. The materials used in the exciplex host were also used as the carrier transport layers to eliminate the energy barriers and thus increase the current density. The hole injection layer structures were varied to examine the hole injection capabilities and the carrier balance. The resulting optimized phosphorescent OLEDs with a broad spectral profile exhibit a 90% coverage ratio in the target ranges from 630 to 690 nm, together with a high peak efficiency of 19.1% (10.2 cd/A and 13.8 lm/W). The proposed device only needs 5.2 V to achieve a power density of 5 mW/cm2, implying that the device could be driven via two series-connected button cell batteries. These results illustrate the feasibility of our design concepts and demonstrate the realization of a portable and lightweight OLED phototherapy light source.

Glucosamine regulation of fibroblast growth factor 21 expression in liver and adipose tissues.

Obesity is associated with metabolic disorders. Fibroblast growth factor 21 (FGF21) has been recognized as important in metabolism. Glucosamine (GLN) has been demonstrated to perform diverse beneficial functions. This study aimed to reveal whether and how GLN would modulate FGF21 production in relation to metabolism. With in vivo model of normal diet (ND) and high-fat diet (HFD) mice receiving GLN injection and in vitro model of mouse AML12 liver cells and differentiated 3T3L1 adipocytes challenged with GLN, GLN appeared to improve the glucose metabolism in HFD and ND mice and to elevate FGF21 protein expression in HFD liver and to increase both FGF21 protein and mRNA levels in WAT from HFD and ND mice and it also upregulated FGF21 expression in both AML12 and differentiated 3T3L1 cells. By using inhibitors against various signaling pathways, p38, Akt, NF-κB, and PKA appeared potentially involved in GLN-mediated FGF21 production in AML12 cells; GLN was able to mediate activation of NF-κB, p38 or PKA/CREB signaling. Our accumulated findings suggest that GLN may potentially improve the metabolic performance by inducing FGF21 production in liver and adipose tissues and such induction in liver cells may act in part due to GLN induction of the NF-κB, p38 and PKA pathways.

Associations of Vitamin D Receptor Single Nucleotide Polymorphisms with Susceptibility to Systemic Sclerosis.

BACKGROUND: This study aims to evaluate whether the Vitamin D receptor (VDR) gene polymorphisms were associated with systemic sclerosis (SSc) in a Chinese Han population. METHODS: Using a hospital-based case-control study including 100 SSc patients and 100 healthy controls. Single nucleotide polymorphisms (SNPs) in the VDR region were genotyped by the improved multiplex ligase detection reaction (i MLDR) method. Haplotypes were also constructed after linkage disequilibrium (LD) analysis. RESULTS: Eight SNPs (rs731236 (TaqI), rs2228570 (FokI), rs7975232 (ApaI), rs1544410 (BsmI), rs11574010 (Cdx2), rs739837 (BglI), rs757343 (Tru9I) and rs11168267) were included. There were significant differences between SSc patients and healthy individuals in ApaI and BglI genotype (both adjusted p = 0.008). Through the genotyping, significantly association of SSc were found for: dominant model of ApaI and BglI (both OR (95% CI) = 1.80 (1.03,3.16), p = 0.040). Furthermore, the elevation of erythrocyte sedimentation rate (ESR) had a higher percentage of BglI GT genotype frequency (p = 0.034) and dominant model of ApaI (p = 0.016) in SSc. There was high linkage disequilibrium was detected in BglI and ApaI polymorphisms (r2 = 1.0, D' = 1.0), Tru9I and rs11168267 (r2 = 0.926, D' = 0.969), respectively. No significant difference were found in these four haplotypes (all p >0.05). The correlation between VD levels and VDR gene polymorphisms was not statistically significant. CONCLUSIONS: Our preliminary study indicates the ApaI and BglI genotype may possibly have a role in the pathogenesis of SSc patients. Dominant model of ApaI and BglI GT genotype frequency may be associated with the increased risk of ESR.

The association between reproductive factors and systemic sclerosis in Chinese women: A case-control study and meta-analysis.

AIM: To examine the associations between female menstrual or reproductive factors and the development of systemic sclerosis (SSc) in China. METHODS: In this hospital-based case-control study, for each subject, data on reproductive and menstrual factors such as number of births, abortions, and age at menarche were obtained by structured questionnaire. Risk estimates, measured by the odds ratio (OR) and 95% confidence interval (CI), were obtained by unconditional logistics regression. Furthermore, meta-analysis was performed and pooled OR with 95% CI for the number of pregnancies and abortions were calculated. RESULTS: There were 166 SSc and 392 female controls seen during the study period. The results showed women with late menarche age (≥17 years) were less likely than those with earlier age at menarche to develop SSc (OR 0.347, 95% CI 0.174-0.693) and compared with women without abortion, women with abortion (1 time) were at reduced risk of developing SSc (P = .036). After adjusting for potential confounders such as occupation and body mass index (BMI), late age at menarche (≥17 years) was associated with a decreased risk of SSc (OR 0.187, 95% CI 0.068-0.513), but abortions were not significantly related to SSc. The meta-analysis revealed there was no association between SSc and abortions or number of pregnancies. No significant publication bias was observed (P > .05). CONCLUSION: Late age at menarche was associated with a reduced risk of SSc but abortion may not be an independent risk factor for SSc. Further investigations are required to verify our findings.

UBASH3A gene polymorphisms and expression profile in rheumatoid arthritis.

OBJECTIVES: Recent evidence has demonstrated that UBASH3A play a pivotal role in multiple autoimmune diseases. In this study, we explored the association between UBASH3A gene single-nucleotide polymorphisms (SNPs) and rheumatoid arthritis (RA) in a Chinese Han population. We also comparatively evaluated the UBASH3A expression profile in peripheral blood mononuclear cells (PBMCs) from patients with RA and healthy controls. METHODS: Four UBASH3A polymorphisms (rs1893592, rs11203203, rs2277798, and rs3788013) were studied in 553 patients with RA and 587 controls in a Chinese population. Genotyping was performed using the Fluidigm 192.24 Dynamic Array Integrated Fluidic Circuit (IFC). For gene expression study, UBASH3A mRNA levels of 30 RA patients and 31 healthy individuals were assessed by real-time quantitative polymerase chain reaction (RT-qPCR). Data were analyzed by SPSS 19.0 software. RESULTS: A significant association between rs1893592 polymorphism and RA was found under all genetic models (all p<.05). We also discovered a significant association between rs3788013 polymorphism and RA in the allele and genotype distributions, as well as the recessive model (all p<.05). Moreover, we found the genotype distribution and allele frequency of rs1893592 were significantly associated with RF phenotype in the RA patients (χ2 = 6.786, p=.034; χ2 = 4.534, p=.033; respectively). We also found the genotype distribution and allele frequency of rs2277798 were significantly associated with anti-CCP phenotype in the RA patients (χ2 = 7.873, p=.020; χ2 = 4.473, p=.034; respectively). However, we did not detect any significant associations between rs11203203 and RA susceptibility and autoantibody profiles (all p>.05). The mRNA expression of UBASH3A was increased in PBMCs of patients with RA when compared to healthy controls (p=.001). CONCLUSIONS: Our observations suggested that the dysregulation of UBASH3A might be associated with the pathogenesis of RA, and UBASH3A gene polymorphisms (rs1893592 and rs3788013) might contribute to RA susceptibility in Chinese Han population.

Association between VDR polymorphisms and multiple sclerosis: systematic review and updated meta-analysis of case-control studies.

Vitamin D receptor (VDR) polymorphisms have been inconsistently investigated in multiple sclerosis (MS). However, published studies demonstrated differences concerning design and effect size. A meta-analysis is necessary to determine the magnitude of the association between VDR polymorphisms and MS risk. The aim of the current study was to quantify the magnitude of the association between BsmI, FokI, ApaI, and TaqI VDR polymorphisms and MS risk. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, we conducted a systematic search and meta-analysis of the VDR gene polymorphisms and the risk of MS. The pooled odds ratios (OR) and 95% confidence interval (CI) were calculated by using Stata Version 11.0 with dominant and recessive models and allele analyses. A total of 4013 cases and 4218 controls in 24 case-control studies were included in the meta-analyses. The results did not indicate an association between any of the VDR polymorphisms and the risk of MS among overall populations, Asians, and Caucasians. However, our subgroup analysis suggests that the A allele was associated with MS risk in Asian populations (P = 0.005, OR = 1.267, 95% CI 1.074-1.496). Interestingly, the sensitivity analysis excluding studies with controls not in HWE showed insignificant association between the A allele and MS risk (P = 0.211), which was different from those in the non-sensitivity analysis. Our preliminary results indicate the VDR gene ApaI, BsmI, FokI, and TaqI polymorphisms may not be associated with elevated MS risk among overall populations. But ApaI polymorphism may confer different susceptibility to MS among different populations, and more well-designed studies with a large sample size are still needed to validate our results.

Association of glucocorticoid receptor gene polymorphisms with systemic lupus erythematosus in a Chinese population.

OBJECTIVES: To identify the association of glucocorticoid receptor (GR) gene polymorphism with systemic lupus erythematosus (SLE). METHODS: A case-control study was carried out, in which 400 Chinese patients with SLE and 400 normal people were enrolled. DNA was extracted using a genomic DNA extraction kit, and tagged single nucleotide polymorphisms (SNPs) were identified by Haploview (4.0) Project from the Chinese HapMap Project. Eighteen tagged SNPs of the GR gene were genotyped by the Multiplex SNaPshot technique. RESULTS: Two GR gene SNPs were associated with the pathogenesis of SLE: rs6865292 (dominant model: crude odds ratio [OR] = 1.526, 95% CI: 1.151-2.025, P = 0.003; adjusted OR = 1.525, 95% CI: 1.149-2.023, P = 0.004; PBH = 0.036) and rs9324921 (dominant model: crude OR = 1.556, 95% CI: 1.173-2.062, P = 0.002; adjusted OR = 1.553, 95% CI: 1.171-2.060, P = 0.002; PBH = 0.036). The haplotype analysis of GR gene SNPs manifested that the haplotype of 'CCGGG' (OR = 2.701, 95% CI: 1.348-5.410, P = 0.004; PBH = 0.036) was a risk factor for the development of SLE. A lower frequency of A-allele of SNP rs4607376 (P = 0.021; OR = 0.794, 95% CI: 0.652-0.966, PBH = 0.126), higher frequency of C-allele of SNP rs6865292 (P = 0.019, OR = 1.317, PBH = 0.126) and A-allele of SNP rs9324921 (P = 0.019, OR = 1.317, PBH = 0.126) may be risk factors for developing SLE. The rs7719514 (recessive model: crude P = 0.044; adjusted P = 0.044, PBH = 0.264), rs7701443 (recessive model: crude P = 0.044, adjusted P = 0.045; PBH = 0.264), rs4607376 (recessive model: crude P = 0.027; adjusted P = 0.026; PBH = 0.264) and haplotype 'CAGCG' (P = 0.044; PBH = 0.198) showed marginal association with the pathogenesis of SLE. In the case group, there were no significant differences between non-lupus nephritis and lupus nephritis. Further, we found that the SNP rs12054797 (F = 3.228, P = 0.041, PBH = 0.342), rs2963156 (F = 3.163, P = 0.043, PBH = 0.342) might be marginally associated with disease activity. CONCLUSIONS: The study indicates that GR genetic polymorphisms may play a major role in the pathogenesis and development of SLE.

Serum adipokines levels in patients with systemic sclerosis: A meta-analysis.

BACKGROUND: Systemic sclerosis is an chronic inflammatory autoimmune diseases. Adipokine has been reported to play an important role in modulating immune responses. Recent studies suggest that adipokine also plays some roles in the pathogenesis of systemic sclerosis (SSc). However, published data regarding the relationship between plasma/serum adipokine levels and SSc are contradictory. The aim of this study was at performing a meta-analysis to derive a more accurate estimation and further investigate the association of plasma/serum leptin and adiponectin levels with SSc patients. METHODS: PubMed, and Web of Science databases (up to Feb 20, 2016) were used to obtain all relative published literatures. The study quality was assessed by the Newcastle-Ottawa scale. Pooled standard mean difference (SMD) with 95% confidence interval (CI) was calculated by random-effect model analysis. RESULTS: A total of fourteen studies were finally included in this meta-analysis. Among them, six of which were studied for the serum adiponectin levels in SSc patients, six of which were studied for the serum leptin levels in SSc patients, and two of them were studied both for serum adiponectin levels and serum leptin levels in SSc patients. The meta-analysis results showed that the serum adiponectin levels in SSc patients were significantly lower than that in normal controls (SMD = -0.608 ng/ml, 95% CI = -1.029 to -0.186, p = 0.005). However, there were no significant differences in serum leptin levels between SSc patients and healthy controls (SMD = -0.990 ng/ml, 95% CI = -2.340 to 0.359, p = 0.150). The subgroup analysis showed that Asia SSc patients with age less than 50 years old had lower plasma/serum adiponectin levels when compared with controls. CONCLUSION: The serum adiponectin levels, but not serum leptin levels, in SSc patients were significantly lower than that in normal controls.

Adjuvant radiation therapy and survival for adenoid cystic carcinoma of the breast.

PURPOSE: The assess the clinical value of different types of surgical procedures and further analyze the effect of adjuvant radiation therapy (RT) for adenoid cystic carcinoma (ACC) of the breast. METHODS: Patients with ACC of the breast were identified using a population-based national registration database (Surveillance, Epidemiology, and End Results, SEER). The Kaplan-Meier method and Cox regression models were performed to determine the impact of the surgical procedures and adjuvant RT associated with cause-specific survival (CSS) and overall survival (OS). RESULTS: A total of 478 patients with ACC of the breast were identified. The median follow-up was 59 months. The 10-year CSS and OS were 87.5% and 75.3%, respectively. For the Kaplan-Meier analysis, the 5-year CSS were 96.1%, 91.8%, 90.2%, and 94.1% in patients that received lumpectomy + adjuvant RT, lumpectomy alone, mastectomy alone, and mastectomy + adjuvant RT, respectively (p = 0.026). In the multivariate Cox analyses, lumpectomy + adjuvant RT was an independent prognostic factor for CSS and OS. Patients that received lumpectomy + adjuvant RT had better survival rates than patients that underwent lumpectomy only (CSS, p = 0.018; OS, p = 0.031) and mastectomy only (CSS, p = 0.010; OS, p = 0.004). CONCLUSION: ACC of the breast has an excellent prognosis. Breast-conserving surgery is a reasonable alternative for patients with ACC of the breast, and adjuvant RT after lumpectomy improved survival rates.

Prognostic value of lymph node ratio in patients with small-cell carcinoma of the cervix based on data from a large national registry.

OBJECTIVE: To investigate the prognostic value of the lymph node ratio (LNR) in patients with small-cell carcinoma of the cervix (SCCC) after cancer-directed surgery using a population-based national registry (Surveillance Epidemiology and End Results [SEER]). METHODS: We retrospectively reviewed the data of SCCC patients in the SEER database from 1980 to 2012. The prognostic impact of LNR with respect to cause-specific survival (CSS) and overall survival (OS) was analyzed. RESULTS: A total of 118 patients with SCCC were identified. The median follow-up was 30.5 months. All these patients were treated with cancer-directed surgery and lymphadenectomy. Sixty (50.8%) patients had nodal metastases. The median LNR was 0.16 in patients with positive lymph nodes. Univariate analysis showed that prognostic factors such as International Federation of Gynecology and Obstetrics (FIGO) stage, nodal status, LNR, and local treatment modalities affected CSS and OS (P<0.05). Multivariate analysis showed that LNR was an independent prognostic factor for CSS and OS. Patients with a higher LNR had worse CSS (hazard ratio [HR]: 8.832; 95% confidence interval [CI]: 3.762-20.738; P<0.001) and OS (HR: 8.462; 95% CI: 3.613-19.821; P<0.001). LNR was associated with CSS and OS by stage, especially in FIGO stage I-II patients. CONCLUSION: LNR is an independent prognostic factor in SCCC patients and it may help to individualize adjuvant therapy.

In vivo immobilization of D-hydantoinase in Escherichia coli.

D-P-Hydroxyphenylglycine (D-HPG) is a precursor required for the synthesis of semi-synthetic antibiotics. This unnatural amino acid can be produced by a transformation reaction mediated by D-hydantoinase (D-HDT) and d-amidohydrolase. In this study, a method was developed to integrate production and immobilization of recombinant D-HDT in vivo. This was approached by first fusion of the gene encoding D-HDT with phaP (encoding phasin) of Ralstonia eutropha H16. The fusion gene was then expressed in the Escherichia coli strain that carried a heterologous synthetic pathway for polyhydroxyalkanoate (PHA). As a result, d-HDT was found to associate with isolated PHA granules. Further characterization illustrated that D-HDT immobilized on PHA exhibited the maximum activity at pH 9 and 60°C and had a half-life of 95 h at 40°C. Moreover, PHA-bound d-HDT could be reused for 8 times with the conversion yield exceeding 90%. Overall, it illustrates the feasibility of this approach to facilitate in vivo immobilization of enzymes in heterologous E. coli strain, which may open a new avenue of enzyme application in industry.

Impact of environmental factors on efficacy of glucocorticoids in Chinese population with systemic lupus erythematosus.

Although glucocorticoids (GCs) are effective in inducing remission in systemic lupus erythematosus (SLE) patients, there is a significant variation in response to therapeutic GCs, and some patients do not achieve full remission. The aim of this study was to explore the impact of environmental factors on the efficacy of GCs in a Chinese population with SLE. This was a prospective cohort study, and a total of 260 SLE patients treated with GCs (prednisone) were followed up for 12 weeks. The efficacy of GCs was measured with the scores on SLE disease activity index. Environmental factors were collected using a questionnaire. Single-variable analysis and multivariate logistic regression analysis were used to discriminate the impact of environmental factors on the efficacy of GCs. Two hundred forty-seven patients (95.00 %) completed the 12-week follow-up. Among these patients, 131 (53.04 %) were classified into sensitive group and 116 (46.96 %) were classified into insensitive group. Results from logistic analysis showed that the following environmental factors were significantly associated with decreased efficacy of GCs: high salt intake (OR = 3.464, 95%CI = 1.481-8.102, P = 0.004), introverted personality (OR = 3.550, 95%CI = 1.901-6.628, P < 0.0001), experience with negative life events (OR = 5.526, 95%CI = 1.612-18.946, P = 0.007), and history of allergy (OR = 2.966, 95%CI = 1.312-6.704, P = 0.009). These results indicate that environmental factors, including salt intake, personality, experience with negative life events, and history of allergy, may play an important role in the efficacy of GCs in the Chinese population with SLE.

Effects of different substrate composition on biosynthesis of polyhydroxybutyrate-co-hydroxyvalerate by recombinant Escherichia coli.

Cupriavidus necator is well known for its ability to accumulate polyhydroxybutyrate (PHB). When supplemented with propionic acid (or sodium propionate) in the growth medium, the bacterium is also able to synthesize polyhydroxybutyrate-co-hydroxyvalerate (PHBV). In order to increase the fraction of 3-hydroxyvalerate (3HV) in PHBV, we cloned the propionate permease gene prpP from C. necator and the propionyl-CoA synthase gene prpE from Cupriavidus taiwanensis and transformed into an Escherichia coli containing phaCAB operon of C. necator. The effects on PHBV accumulation in cells co-expressed with phaCAB and prpE or prpP in the media contained mixed carbon sources (glucose and sodium propionate) were evaluated. The HV fraction in PHBV increased when prpE or prpP was overexpressed in the cells. Concentrations of yeast extracts could also affect the fraction of HV. In addition, when glucose was replaced by sodium pyruvate, sodium succinate, or sodium gluconate, only PHB were detected in the recombinant strains.

Marker-free chromosomal expression of foreign and native genes in Escherichia coli.

Genetic manipulation of Escherichia coli strains for desired traits is the most applied strain engineering approach in industrial applications. For chromosomal insertion of genes and controlled expression of genomic genes in E. coli, the replicon-free and markerless method is described based on a series of conditional-replication plasmids called phage-integration vectors. They mainly carry the multiple cloning site and the prophage attachment site, which are sandwiched by two FRT sites. With the aid of the phage integrase from conditional-replication helper plasmids, the passenger genes of either foreign or native type incorporated into the integration vectors can be specifically integrated into bacterial genome at the prophage attachment site. Finally, the inserted DNA containing replicon and/or selective markers in integrants can be eliminated by the act of the FLP recombinase provided from a conditional-replication helper plasmid.

Engineering of Escherichia coli for targeted delivery of transgenes to HER2/neu-positive tumor cells.

Targeting of non-phagocytic tumor cells and prompt release of gene cargos upon entry into tumors are two limiting steps in the bacterial gene delivery path. To tackle these problems, the non-pathogenic Escherichia coli strain BL21(DE3) was engineered to display the anti-HER2/neu affibody on the surface. After co-incubation with tumor cells for 3 h, the anti-HER2/neu affibody-presenting E. coli strain was selectively internalized into HER2/neu-positive SKBR-3 cells. The invasion efficiency reached as high as 30%. Furthermore, the bacteria were equipped with the phage ϕX174 lysin gene E-mediated autolysis system. Carrying the transgene (e.g., eukaryotic green fluorescent protein, GFP), the tumor-targeting bacteria were subjected to the thermal shock to trigger the autolysis system upon entry into HER2/neu-positive cells. Flow cytometric analysis revealed that 3% of infected cells expressed GFP 24 h post thermal induction. Overall, the results show a promise of the proposed approach for developing bacteria as a delivery carrier.

Production and characterization of ectoine by Marinococcus sp. ECT1 isolated from a high-salinity environment.

A halophilic bacterium isolated from a salt environment in southern Taiwan was identified as a Marinococcus sp. ECT1. This bacterium could synthesize and accumulate intracellular ectoine as a compatible solute capable of resisting osmotic stress in a hyper-osmotic environment. This study also developed a semi-synthesized medium (YAMS medium), capable of facilitating the growth of this Marinococcus sp. ECT1 with 600 mg/L crude ectoine production. Moreover, Marinococcus sp. ECT1 was grown on YAMS medium containing different initial yeast extract concentrations (C(YE)) (0 to 60 g/L) to demonstrate how C(YE) affects crude ectoine production. While the maximum cell concentration was increased by 23-fold when the C(YE) was 40 g/L, the maximum crude ectoine production reached 2.5 g/L when C(YE) was 40 g/L. In addition to demonstrating the success of the fermentation strategy of ectoine in increasing the production and production yield, experimental results further demonstrated that the fermentation medium of ectoine is highly promising for commercialization. Furthermore, the molecular weight and chemical structure of ectoine were identified and characterized by FAB-MS and (1)H-NMR.