Polycomb group (PcG) proteins prevent the assembly of abnormal synaptonemal complex structures during meiosis.

The synaptonemal complex (SC) is a proteinaceous scaffold that is assembled between paired homologous chromosomes during the onset of meiosis. Timely expression of SC coding genes is essential for SC assembly and successful meiosis. However, SC components have an intrinsic tendency to self-organize into abnormal repetitive structures, which are not assembled between the paired homologs and whose formation is potentially deleterious for meiosis and gametogenesis. This creates an interesting conundrum, where SC genes need to be robustly expressed during meiosis, but their expression must be carefully regulated to prevent the formation of anomalous SC structures. In this manuscript, we show that the Polycomb group protein Sfmbt, the Drosophila ortholog of human MBTD1 and L3MBTL2, is required to avoid excessive expression of SC genes during prophase I. Although SC assembly is normal after Sfmbt depletion, SC disassembly is abnormal with the formation of multiple synaptonemal complexes (polycomplexes) within the oocyte. Overexpression of the SC gene corona and depletion of other Polycomb group proteins are similarly associated with polycomplex formation during SC disassembly. These polycomplexes are highly dynamic and have a well-defined periodic structure. Further confirming the importance of Sfmbt, germ line depletion of this protein is associated with significant metaphase I defects and a reduction in female fertility. Since transcription of SC genes mostly occurs during early prophase I, our results suggest a role of Sfmbt and other Polycomb group proteins in downregulating the expression of these and other early prophase I genes during later stages of meiosis.

Implementation of a novel computer assisted telephone follow-up model for older patients after emergency department discharge in an Asian population.

BACKGROUND: While the impact of telephone follow-up (TFU) for older emergency department (ED) patients is controversial, its effects on the Asian population remain uncertain. In this study, we evaluated the effectiveness of a novel computer assisted TFU model specifically for this demographic. METHODS: At a Taiwanese tertiary medical center, we developed a TFU protocol that included a referral and case management system within the ED hospital information system. We provided TFU to older discharged patients between April 1, 2021, and May 31, 2021. We compared this cohort with a non-TFU cohort of older ED patients and analyzed demographic characteristics and post-ED discharge outcomes. RESULTS: The TFU model was successfully implemented, with 395 patients receiving TFU and 191 without TFU. TFU patients (median age: 76 years, male proportion: 48.9%) differed from non-TFU patients (median age: 74 years, male proportion: 43.5%). Compared with the non-TFU cohort, the multivariate logistic regression analysis revealed that the TFU cohort had a lower total medical expenditure < 1 month (adjusted odds ratio [AOR]: 0.32; 95% CI: 0.21 - 0.47 for amounts exceeding 5,000 New Taiwan Dollars), and higher satisfaction (AOR: 2.80; 95% CI: 1.46 - 5.36 for scores > 3 on a five-point Likert Scale). However, the TFU cohort also had a higher risk of hospitalization < 1 month (AOR: 2.50; 95% CI: 1.31 - 4.77) compared to the non-TFU cohort. CONCLUSION: Computer-assisted TFU appears promising. Further research involving a larger number of patients and validation in other hospitals is necessary to bolster the evidence and extend the findings to a broader context.

A Novel Cross-Sensor Transfer Diagnosis Method with Local Attention Mechanism: Applied in a Reciprocating Pump.

Data-driven mechanical fault diagnosis has been successfully developed in recent years, and the task of training and testing data from the same distribution has been well-solved. However, for some large machines with complex mechanical structures, such as reciprocating pumps, it is often not possible to obtain data from specific sensor locations. When the sensor position is changed, the distribution of the features of the signal data also changes and the fault diagnosis problem becomes more complicated. In this paper, a cross-sensor transfer diagnosis method is proposed, which utilizes the sharing of information collected by sensors between different locations of the machine to complete a more accurate and comprehensive fault diagnosis. To enhance the model's perception ability towards the critical part of the fault signal, the local attention mechanism is embedded into the proposed method. Finally, the proposed method is validated by applying it to experimentally acquired vibration signal data of reciprocating pumps. Excellent performance is demonstrated in terms of fault diagnosis accuracy and sensor generalization capability. The transferability of practical industrial faults among different sensors is confirmed.

Design and Analysis of a Cardioid Flow Tube Valveless Piezoelectric Pump for Medical Applications.

Piezoelectric pumps play an important role in modern medical technology. To improve the flow rate of valveless piezoelectric pumps with flow tube structures and promote the miniaturization and integration of their designs, a cardioid flow tube valveless piezoelectric pump (CFTVPP) is proposed in this study. The symmetric dual-bend tube design of CFTVPP holds great potential in applications such as fluid mixing and heat dissipation systems. The structure and working principle of the CFTVPP are analyzed, and flow resistance and velocity equations are established. Furthermore, the flow characteristics of the cardioid flow tube (CFT) are investigated through computational fluid dynamics, and the output performance of valveless piezoelectric pumps with different bend radii is studied. Experimental results demonstrate that CFTVPP exhibits the pumping effect, with a maximum vibration amplitude of 182.5 µm (at 22 Hz, 100 V) and a maximum output flow rate of 5.69 mL/min (at 25 Hz, 100 V). The results indicate that a smaller bend radius of the converging bend leads to a higher output flow rate, while the performance of valveless piezoelectric pumps with different diverging bends shows insignificant differences. The CFTVPP offers advantages such as a high output flow rate, low cost, small size for easy integration, and ease of manufacturing.

Interdisciplinary collaboration and computer-assisted home healthcare referral in the emergency department: a retrospective cohort study.

AIM: Home healthcare (HHC) provides continuous care for disabled patients. However, HHC referral after the emergency department (ED) discharge remains unclear. Thus, this study aimed its clarification. METHODS: A computer-assisted HHC referral by interdisciplinary collaboration among emergency physicians, case managers, nurse practitioners, geriatricians, and HHC nurses was built in a tertiary medical center in Taiwan. Patients who had HHC referrals after ED discharge between February 1, 2020 and September 31, 2020, were recruited into the study. A non-ED HHC cohort who had HHC referrals after hospitalization from the ED was also identified. Comparison for clinical characteristics and uses of medical resources was performed between ED HHC and non-ED HHC cohorts. RESULTS: The model was successfully implemented. In total, 34 patients with ED HHC and 40 patients with non-ED HHC were recruited into the study. The female proportion was 61.8% and 67.5%, and the mean age was 81.5 and 83.7 years in ED HHC and non-ED HHC cohorts, respectively. No significant difference was found in sex, age, underlying comorbidities, and ED diagnoses between the two cohorts. The ED HHC cohort had a lower median total medical expenditure within 3 months (34,030.0 vs. 56,624.0 New Taiwan Dollars, p = 0.021) compared with the non-ED HHC cohort. Compared to the non-ED HHC cohort, the ED HHC cohort had a lower ≤ 1 month ED visit, ≤ 6 months ED visit, and ≤ 3 months hospitalization; however, differences were not significant. CONCLUSION: An innovative ED HHC model was successfully implemented. Further studies with more patients are warranted to investigate the impact.

Computerized tool and interdisciplinary care for older patients with delirium in the emergency department: a novel model in Taiwan.

AIMS: A computerized tool and interdisciplinary care were implemented to develop a novel model for older patients with delirium in the emergency department (ED). METHODS: We developed a computerized tool using a delirium triage screen and brief confusion assessment in the hospital information system, performed education for the healthcare providers, and developed a continuous care protocol. Comparisons for outcomes between pre- and post-intervention periods were performed. RESULTS: Compared with the pre-intervention period, patients in the post-intervention period had shorter hospitalization stay, lower expenditure of hospitalization, more likely to return home, lower ED revisits of ≤ 3 days, re-hospitalization of ≤ 14 days, and mortality of ≤ 1 month. All mentioned differences were not statistically significant. CONCLUSIONS: A novel model was successfully developed for delirium management in older patients in the ED. Outcome differences were not significant; however, the result is promising, which gives us an important reference in the future.

Effect of High-Frequency Repetitive Peripheral Magnetic Stimulation on Motor Performance in Intracerebral Haemorrhage: A Clinical Trial.

OBJECTIVES: The aim of the randomized, double-blind, sham-controlled trial was to explore the efficacy and safety of HF-rPMS synchronosly applied to the axilla (stimulating the brachial plexus) and the popliteal fossa (stimulating the tibial nerve and common peroneal nerve) in patients with intracerebral hemorrhage on rehabilitation of motor functions. MATERIALS AND METHODS: Patients with intracerebral haemorrhage in the early period were recruited and randomly assigned to the HF-rPMS group or the sham rPMS group. The two synchrous coils of magnetic stimulation in the two groups were respectively applied to the axilla and the popliteal fossa of the affected limb. But the sham group received the ineffective rPMS and only heard the sound as occured in the HF-rPMS group. Clinical outcomes included the change of Fugl-Meyer Assessment (FMA) scale and Medical Research Council (MRC) scale before and after HF-rPMS. RESULTS: Of 76 eligible patients, 30 were included and only 26 patients completed this study. The diferences on the improvement of the upper extremity FMA (P=0.012), the lower extremity FMA (P=0.001), the proximal MRC of upper extremity (p = 0.043), the proximal MRC of lower extremity (p= 0.004) and the distal MRC scores of lower extremity (p= 0.008) between the the HF-rPMS group and sham rPMS group were statistically signifcant. CONCLUSIONS: Synchrous HF-rPMS intervention at the axilla and the popliteal fossa significantly improved motor function and proximal muscle strength of upper and lower limb of patients in acute or early subacute phase of intracerebral hemorrhage.

Associations between perceived stress and BMI and waist circumference in Chinese adults: data from the 2015 China Health and Nutrition Survey.

OBJECTIVES: To assess the association between perceived stress and adiposity among Chinese adults. DESIGN: Cross-sectional study. Perceived stress was assessed using the 14-item perceived stress scale. Associations between quintiles of perceived stress and BMI and waist circumference were assessed using linear regression models and multinomial regression models. Estimates were adjusted for sociodemographic characteristics. SETTING: 2015 China Health and Nutrition Survey: 12 provinces covering a variety of geographic, economic development and health indicator situations. PARTICIPANTS: A total of 8385 adults of both sexes, aged 18-99 years, were included. RESULTS: Overall, the mean perceived stress score was 22·7 (6·2), mean BMI was 24·3 (3·6) kg/m2 and prevalence of obesity (BMI ≥ 30 kg/m2) was 6·0 %. There were inverse associations between perceived stress quintiles with continuous BMI (P < 0·001), BMI categories (P = 0·015) and waist circumference (P = 0·047). Compared to adults in the lowest quintile of perceived stress, adults in the highest quintile of perceived stress had 0·44 kg/m2 lower mean BMI (95 % CI: -0·67, -0·21), 0·72 times the prevalence of obesity (95 % CI: 0·55, 0·94) and 0·73 times the prevalence of abdominal obesity (95 % CI: 0·61, 0·88). Results were similar when using Chinese-specific cut-points. CONCLUSION: Our results showed inverse associations between perceived stress quintiles and adiposity among Chinese adults. Future studies should aim to better understand the directionality of the observed associations and the potential biological and behavioural mechanisms underlying these associations in the Chinese population.

Predicting outcomes in older ED patients with influenza in real time using a big data-driven and machine learning approach to the hospital information system.

BACKGROUND: Predicting outcomes in older patients with influenza in the emergency department (ED) by machine learning (ML) has never been implemented. Therefore, we conducted this study to clarify the clinical utility of implementing ML. METHODS: We recruited 5508 older ED patients (≥65 years old) in three hospitals between 2009 and 2018. Patients were randomized into a 70%/30% split for model training and testing. Using 10 clinical variables from their electronic health records, a prediction model using the synthetic minority oversampling technique preprocessing algorithm was constructed to predict five outcomes. RESULTS: The best areas under the curves of predicting outcomes were: random forest model for hospitalization (0.840), pneumonia (0.765), and sepsis or septic shock (0.857), XGBoost for intensive care unit admission (0.902), and logistic regression for in-hospital mortality (0.889) in the testing data. The predictive model was further applied in the hospital information system to assist physicians' decisions in real time. CONCLUSIONS: ML is a promising way to assist physicians in predicting outcomes in older ED patients with influenza in real time. Evaluations of the effectiveness and impact are needed in the future.

Validation of shock index for predicting mortality in older patients with dengue fever.

BACKGROUND: Older adults have a higher mortality for dengue fever (DF). However, the best method for predicting mortality is still unclear. AIMS: We conducted this study to evaluate the shock index (SI) for this issue. METHODS: A retrospective case-control study was conducted by recruiting older patients (≥ 65 years old) with DF who visited the study hospital in southern Taiwan during the 2015 DF outbreak. Demographic data, vital signs, past histories, decision groups, complications, and mortality were included in the analyses. We evaluated the accuracy of SI ≥ 1 for predicting 30-day mortality in this population. RESULTS: A total of 626 patients with a mean age of 74.1 years and nearly equal sex distribution were recruited. The mean of SI (± standard deviation [SD]) was 0.6 (± 0.2) and patients with a SI ≥ 1 accounted for 3.5% of the total patients. Logistic regression showed that patients with SI ≥ 1 had a higher mortality than those with SI < 1 (odds ratio: 8.49; 95% confidence interval: 1.76-17.92). The area under the receiver-operating characteristic was 0.76, and the Hosmer-Lemeshow goodness of fit test was 0.48. The SI ≥ 1 had a sensitivity, specificity, positive predictive value, and negative predictive value of 14.8%, 97.0%, 18.2%, and 96.2% for predicting mortality. CONCLUSIONS: The SI ≥ 1 is an easy tool that can be potentially used to predict 30-day mortality in older DF patients, especially in DF outbreak. It has a high specificity and negative predictive value for excluding patients with high-risk mortality.

Reprogramming of sugar transport pathways in Escherichia coli using a permeabilized SecY protein-translocation channel.

In the initial step of sugar metabolism, sugar-specific transporters play a decisive role in the passage of sugars through plasma membranes into cytoplasm. The SecY complex (SecYEG) in bacteria forms a membrane channel responsible for protein translocation. The present work shows that permeabilized SecY channels can be used as nonspecific sugar transporters in Escherichia coli. SecY with the plug domain deleted allowed the passage of glucose, fructose, mannose, xylose, and arabinose, and, with additional pore-ring mutations, facilitated lactose transport, indicating that sugar passage via permeabilized SecY was independent of sugar stereospecificity. The engineered E. coli showed rapid growth on a wide spectrum of monosaccharides and benefited from the elimination of transport saturation, improvement in sugar tolerance, reduction in competitive inhibition, and prevention of carbon catabolite repression, which are usually encountered with native sugar uptake systems. The SecY channel is widespread in prokaryotes, so other bacteria may also be engineered to utilize this system for sugar uptake. The SecY channel thus provides a unique sugar passageway for future development of robust cell factories for biotechnological applications.

Effects of Cd and Pb on diversity of microbial community and enzyme activity in soil.

Pollution due to heavy metals is a serious global environmental problem, particularly in China. It is thus important to study the effects of heavy metal pollution, especially in mining areas. Cadmium(Cd) and lead(Pb) severely damage the microbial life in soil. The concentration of heavy metals and their toxic effects on microbes and enzymes in soil were examined in this study using contaminated soil samples. The Biolog method was used to analyze the characteristics of the microbial community. The results showed that the addition of Cd2+ and Pb2+ in different concentrations has a significant impact on microbial and enzyme activity in soil. With an increase in their concentrations, activities of the microbial community and enzymes decreased gradually. Each index related to the structure of the microbial community in soil decreased, indicating that pollution due to Cd and Pb reduced its size and functional activity. This study provides a reference for future research on the functional diversity of the microbial community in soil and plays its role in their environmental management.

[Prescription and medication regularity of traditional Chinese medicine for treating child pneumonia based on data mining].

To analyze and summarize formulae of traditional Chinese medicine(TCM) in the treatment of pediatric pneumonia in CNKI based on the data mining method. CNKI was retrieved for literatures of TCM treatment of pneumonia in children in recent 20 years, which was taken the data source. After screening, a prescription database was established. Frequency analysis, association rules Apriori algorithm and complex system entropy clustering analysis methods integrated in TCMISS(V2.5) were applied in data analysis and mining. Core drugs and their properties and flavors, medication modes and new prescriptions were summarized and studied. A total of 408 prescriptions were screened out, involving 218 drugs, among which Chinese herbal medicines with the highest use frequency included Gancao(Glycyrrhizae Radix et Rhizoma), Xingren(Armeniacae Semen Amarum), Huangqin(Scutellariae Radix), Mahuang(Ephedrae Herba), Jiegeng(Platycodonis Radix), Banxia(Pinelliae Rhizoma), Sangbaipi(Mori Cortex), Chenpi(Citri Reticulatae Pericarpium), Fuling(Poria), Jinyinhua(Japonicae Lonicerae Flos). Flavors were mainly bitter, sweet and pungent; and the main medicinal property was cold. Most of these drugs entered lung meridian(LU), stomach meridian(ST) and spleen meridian(SP); and the association rules between drugs were established. Totally 6 new prescriptions were obtained through entropy hierarchical cluster analysis. Our results objectively presented, at present, TCM focuses on phlegm and heat in treating children's pneumonia, which targets at lung and also treats spleen and stomach. The main therapies aim to clear heat and resolve phlegm, ventilate lung and relieve cough and dyspnea, release exterior and dissipate cold, and relieve cough and eliminate phlegm. The results of statistical analysis are basically consistent with the consensus of experts, which can provide theoretical basis for clinical medication and drug development.

Transcription Factors VND1-VND3 Contribute to Cotyledon Xylem Vessel Formation.

Arabidopsis (Arabidopsis thaliana) VASCULAR-RELATED NAC-DOMAIN1 (VND1) to VND7 encode a group of NAC domain transcription factors that function as master regulators of xylem vessel element differentiation. These transcription factors activate the transcription of genes required for secondary cell wall formation and programmed cell death, key events in xylem vessel element differentiation. Because constitutive overexpression of VND6 and VND7 induces ectopic xylem vessel element differentiation, functional studies of VND proteins have largely focused on these two proteins. Here, we report the roles of VND1, VND2, and VND3 in xylem vessel formation in cotyledons. Using our newly established in vitro system in which excised Arabidopsis cotyledons are stimulated to undergo xylem cell differentiation by cytokinin, auxin, and brassinosteroid treatment, we found that ectopic xylem vessel element differentiation required VND1, VND2, and VND3 but not VND6 or VND7. The importance of VND1, VND2, and VND3 also was indicated in vivo; in the vnd1 vnd2 vnd3 seedlings, xylem vessel element differentiation of secondary veins in cotyledons was inhibited under dark conditions. Furthermore, the light responsiveness of VND gene expression was disturbed in the vnd1 vnd2 vnd3 mutant, and vnd1 vnd2 vnd3 failed to recover lateral root development in response to the change of light conditions. These findings suggest that VND1 to VND3 have specific molecular functions, possibly linking light conditions to xylem vessel formation, during seedling development.

Engineering and manipulation of a mevalonate pathway in Escherichia coli for isoprene production.

Isoprene is a useful phytochemical with high commercial values in many industrial applications including synthetic rubber, elastomers, isoprenoid medicines, and fossil fuel. Currently, isoprene is on large scale produced from petrochemical sources. An efficient biological process for isoprene production utilizing renewable feedstocks would be an important direction of research due to the fossil raw material depletion and air pollution. In this study, we introduced the mevalonate (MVA) pathway genes/acetoacetyl-coenzyme A thiolase (mvaE) and MVA synthase (mvaS) from Enterococcus faecalis (E. faecalis); MVA kinase (mvk) derived from Methanosarcina mazei (M. mazei); and phosphomevalonate kinase (pmk), diphosphomevalonate decarboxylase (mvaD), and isopentenyl diphosphate isomerase (idi) from Streptococcus pneumoniae (S. pneumoniae) to accelerate dimethylallyl diphosphate (DMAPP) accumulation in Escherichia coli (E. coli). Together with a codon-optimized isoprene synthase (ispS) from Populus alba (P. alba), E. coli strain succeeded in formation of isoprene. We then manipulated the heterologous MVA pathway for high-level production of isoprene, by controlling the gene expression levels of the MVA pathway genes. We engineered four E. coli strains which showed different gene expression levels and different isoprene productivities, and we also characterized them with quantitative real-time PCR and metabolite analysis. To further improve the isoprene titers and release the toxicity to cells, we developed the extraction fermentation by adding dodecane in cultures. Finally, strain BL2T7P1TrcP harboring balanced gene expression system produced 587 ± 47 mg/L isoprene, with a 5.2-fold titer improvement in comparison with strain BL7CT7P. This work indicated that a balanced metabolic flux played a significant role to improve the isoprene production via MVA pathway.

Establishing a protocol for water sample processing for the detection of Blastocystis sp.

This study was aimed at establishing a protocol for water sample processing for the detection of Blastocystis sp. using distilled water spiked with Blastocystis sp. cysts. The study established a protocol involving eight technical aspects, namely, storage temperature, storage duration, minimum water sample volume, optimum relative centrifugal force, centrifugation duration, minimum number of cyst for inoculation in Jones' medium and turn-around-time for the detection of vacuolar forms of Blastocystis sp. Results showed a minimum of 1.0 L water sample should be collected and processed on the same day. Otherwise, it should be stored at 4 °C and processed within 3 days. Water sample should be centrifuged at 1400×g for 10 min. For the isolation of Blastocystis sp. cysts, parasite pellet could be layered on top of Ficoll-Paque™ PLUS, centrifuged at 1400×g for 20 min and washed twice using 0.9% saline with centrifugation at 1400×g for 10 min. A minimum of 1 × 105 cysts could then be inoculated in Jones' medium supplement with 10% horse serum, incubated at 37 °C and examined for any presence of vacuolar forms of Blastocystis sp. after 3 days of inoculation. A protocol for water sample processing for the detection of Blastocystis sp. has successfully been established. The protocol was validated using 106 various water samples. This protocol will be very useful in determining the extent of Blastocystis sp. contamination in water sources in order to identify the seriousness of contamination.

Arabidopsis Group IIId ERF proteins positively regulate primary cell wall-type CESA genes.

The cell wall determines morphology and the environmental responses of plant cells. The primary cell wall (PCW) is produced during cell division and expansion, determining the cell shape and volume. After cell expansion, specific types of plant cells produce a lignified wall, known as a secondary cell wall (SCW). We functionally analyzed Group IIId Arabidopsis AP2/EREBP genes, namely ERF34, ERF35, ERF38, and ERF39, which are homologs of a rice ERF gene previously proposed to be related to SCW biosynthesis. Expression analysis revealed that these four genes are expressed in regions related to cell division and/or cell differentiation in seedlings (i.e., shoot apical meristems, the primordia of leaves and lateral roots, trichomes, and central cylinder of primary roots) and flowers (i.e., vascular tissues of floral organs and replums and/or valve margins of pistils). Overexpression of ERF genes significantly upregulated PCW-type, but not SCW-type, CESA genes encoding cellulose synthase catalytic subunits in Arabidopsis seedlings. Transient co-expression reporter analysis indicated that ERF35, ERF38, and ERF39 possess transcriptional activator activity, and that ERF34, ERF35, ERF38, and ERF39 upregulated the promoter activity of CESA1, a PCW-type CESA gene, through the DRECRTCOREAT elements, the core cis-acting elements known to be recognized by AP2/ERF proteins. Together, our findings show that Group IIId ERF genes are positive transcriptional regulators of PCW-type CESA genes in Arabidopsis and are possibly involved in modulating cellulose biosynthesis in response to developmental requirements and environmental stimuli.

Biosynthesis of D-glucaric acid from sucrose with routed carbon distribution in metabolically engineered Escherichia coli.

D-glucaric acid is a promising platform compound used to synthesize many other value-added or commodity chemicals. The engineering of Escherichia coli for efficiently converting D-glucose to D-glucaric acid has been attempted for several years, with mixed sugar fermentation recently gaining growing interests due to the increased D-glucaric acid yield. Here, we co-expressed cscB, cscA, cscK, ino1, miox, udh, and suhB in E. coli BL21 (DE3), functionally constructing an unreported route from sucrose to D-glucaric acid. Further deletion of chromosomal zwf, pgi, ptsG, uxaC, gudD, over-expression of glk, and use of a D-fructose-dependent translation control system for pgi enabled the strain to use sucrose as the sole carbon source while achieving a high product titer and yield. The titer of D-glucaric acid in M9 medium containing 10 g/L sucrose reached ~1.42 g/L, with a yield of ~0.142 g/g on sucrose.

Metabolic engineering of Escherichia coli for the synthesis of polyhydroxyalkanoates using acetate as a main carbon source.

BACKGROUND: High production cost of bioplastics polyhydroxyalkanoates (PHA) is a major obstacle to replace traditional petro-based plastics. To address the challenges, strategies towards upstream metabolic engineering and downstream fermentation optimizations have been continuously pursued. Given that the feedstocks especially carbon sources account up to a large portion of the production cost, it is of great importance to explore low cost substrates to manufacture PHA economically. RESULTS: Escherichia coli was metabolically engineered to synthesize poly-3-hydroxybutyrate (P3HB), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) using acetate as a main carbon source. Overexpression of phosphotransacetylase/acetate kinase pathway was shown to be an effective strategy for improving acetate assimilation and biopolymer production. The recombinant strain overexpressing phosphotransacetylase/acetate kinase and P3HB synthesis operon produced 1.27 g/L P3HB when grown on minimal medium supplemented with 10 g/L yeast extract and 5 g/L acetate in shake flask cultures. Further introduction succinate semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, and CoA transferase lead to the accumulation of P3HB4HB, reaching a titer of 1.71 g/L with a 4-hydroxybutyrate monomer content of 5.79 mol%. When 1 g/L of α-ketoglutarate or citrate was added to the medium, P3HB4HB titer increased to 1.99 and 2.15 g/L, respectively. To achieve PHBV synthesis, acetate and propionate were simultaneously supplied and propionyl-CoA transferase was overexpressed to provide 3-hydroxyvalerate precursor. The resulting strain produced 0.33 g/L PHBV with a 3-hydroxyvalerate monomer content of 6.58 mol%. Further overexpression of propionate permease improved PHBV titer and 3-hydroxyvalerate monomer content to 1.09 g/L and 10.37 mol%, respectively. CONCLUSIONS: The application of acetate as carbon source for microbial fermentation could reduce the consumption of food and agro-based renewable bioresources for biorefineries. Our proposed metabolic engineering strategies illustrate the feasibility for producing polyhydroxyalkanoates using acetate as a main carbon source. Overall, as an abundant and renewable resource, acetate would be developed into a cost-effective feedstock to achieve low cost production of chemicals, materials, and biofuels.

A novel DMAPP-responding genetic circuit sensor for high-throughput screening and evolving isoprene synthase.

High-throughput screening is a popular tool for collating biological data which would otherwise require the use of excessive resources. In this study, an artificial genetic circuit sensor responding to dimethylallyl diphosphate (DMAPP) was constructed based on a modified L-arabinose operon for high-throughput screening and isoprene synthase (ispS) evolution in Escherichia coli (E. coli). As a first step, the DNA sequence of the L-arabinose ligand-binding domain (LBD) was replaced with an ispS gene to enable the AraC operon responding to DMAPP, which is the substrate of the IspS enzyme. Then, an enhanced GFP (eGFP) was also introduced as a reporter for pBAD promoter. The expression level of the reporter was monitored using either of the two tools: flow cytometer (FCM) and microplate reader. Sequentially, we observed that a high DMAPP concentration led to low eGFP fluorescence, and the overexpression of ispS gene, which consumes DMAPP, resulted in a high eGFP expression. These results demonstrated that the artificial genetic circuit sensor responded directly to the intracellular concentration of DMAPP, and the expression of IspS enzyme could be positively correlated to the expression level of eGFP. Finally, we identified two IspS mutants with different activities from an ispS gene library and further validated the screening method.