Targets For Further Improvement Research Articles

C-FOS is an integral component of the IKZF1 transactivator complex and mediates lenalidomide resistance in multiple myeloma.

The immunomodulatory drug lenalidomide, which is now widely used for the treatment of multiple myeloma (MM), exerts pharmacological action through the ubiquitin-dependent degradation of IKZF1 and subsequent down-regulation of interferon regulatory factor 4 (IRF4), a critical factor for the survival of MM cells. IKZF1 acts principally as a tumour suppressor via transcriptional repression of oncogenes in normal lymphoid lineages. In contrast, IKZF1 activates IRF4 and other oncogenes in MM cells, suggesting the involvement of unknown co-factors in switching the IKZF1 complex from a transcriptional repressor to an activator. The transactivating components of the IKZF1 complex might promote lenalidomide resistance by residing on regulatory regions of the IRF4 gene to maintain its transcription after IKZF1 degradation. To identify unknown components of the IKZF1 complex, we analyzed the genome-wide binding of IKZF1 in MM cells using chromatin immunoprecipitation-sequencing (ChIP-seq) and screened for the co-occupancy of IKZF1 with other DNA-binding factors on the myeloma genome using the ChIP-Atlas platform. We found that c-FOS, a member of the activator protein-1 (AP-1) family, is an integral component of the IKZF1 complex and is primarily responsible for the activator function of the complex in MM cells. The genome-wide screening revealed the co-occupancy of c-FOS with IKZF1 on the regulatory regions of IKZF1-target genes, including IRF4 and SLAMF7, in MM cells but not normal bone marrow progenitors, pre-B cells or mature T-lymphocytes. c-FOS and IKZF1 bound to the same consensus sequence as the IKZF1 complex through direct protein-protein interactions. The complex also includes c-JUN and IKZF3 but not IRF4. Treatment of MM cells with short-hairpin RNA against FOS or a selective AP-1 inhibitor significantly enhanced the anti-MM activity of lenalidomide in vitro and in two murine MM models. Furthermore, an AP-1 inhibitor mitigated the lenalidomide resistance of MM cells. C-FOS determines lenalidomide sensitivity and mediates drug resistance in MM cells as a co-factor of IKZF1 and thus, could be a novel therapeutic target for further improvement of the prognosis of MM patients.

Patients’ and health care providers’ perspectives on quality of hemophilia care in the Netherlands: a questionnaire and interview study

BackgroundHemophilia care has improved greatly because of advances in treatment options and comprehensive care. In-depth insight into the perspectives of persons with hemophilia and health care providers on their care may provide targets for further improvements. ObjectivesTo assess satisfaction of the hemophilia population with their care, to explore factors determining care satisfaction, and to identify areas for potential health care improvements, including digital health tools. MethodsFirst, to assess care satisfaction and factors determining satisfaction and health care improvements, data from a nationwide, cross-sectional questionnaire among 867 adult and pediatric Dutch persons with hemophilia A or B were analyzed. This included the Hemophilia Patient Satisfaction Scale questionnaire, Canadian Hemophilia Outcomes Kids’ Life Assessment Tool satisfaction questions, a visual analog scale satisfaction score, and open questions. Second, to further explore factors determining satisfaction and health care improvements, semistructured interviews were conducted with 19 persons with hemophilia or their parents and 18 health care providers. ResultsHigh care satisfaction was found, with an overall median Hemophilia Patient Satisfaction Scale score of 12 (IQR, 6-21). Participants in the interviews reported that patient-professional interactions, availability of care, and coordination of care were major factors determining satisfaction. Suggested health care improvements included improved information provision and coordination of care, especially shared care with professionals not working within comprehensive care centers. Participants suggested that digital health tools could aid in this. ConclusionSatisfaction with hemophilia care is high among persons with hemophilia in the Netherlands, although several potential improvements have been identified. Accentuating these is especially relevant in the current era of treatment innovations, in which we might focus less on other aspects of care.

Assessment of antibiotic prescribing patterns at dental and primary health care clinics according to WHO Access, Watch, Reserve (AWaRe) classification

BackgroundThe 2019 WHO Access, Watch, Reserve (AWaRe) antibiotic classification framework aims to prevent irrational prescribing of antibiotics used to treat widespread infections. This study explored antibiotic prescribing pattern for appropriate indications by family physicians and general dentists in primary health care practices. MethodsA retrospective review of patients’ electronic medical records was conducted over 6 months, from May 1, 2020, to November 30, 2020. The data were collected from 24 general family medicine and dental practices within the North West Armed Forces in Tabuk city. Antibiotic prescribing for systemic use (J01) was assessed by the number of prescriptions and the number defined daily doses (DDDs) and then analyzed according to the AWaRe classification. The prescribing of antibiotics for appropriate indications was assessed through comparing the prescription pattern with the recently published and relevant clinical guidelines. Multivariate logistic regression analysis was used to predict the association between the prescribing of AWaRe category and some demographic and disease-related factors. ResultsIn total, 752 prescriptions of antibiotics were collected. Watch-group antibiotics such as second-generation cephalosporin and macrolides were more likely prescribed (51.1%) based on the number of prescriptions and (52.2%) based on DDDs compared with Access-group antibiotics (48.9%) and (47.8%), respectively. The percentages of Watch group antibiotics for children and adults were 66.7% and 42.9%, respectively. Adherence to prescribing guidelines was poor for children (27.2%) and adults (64%). Being a child (adjusted odds ratio [OR]: 2.89; 95% confidence interval [CI] = 1.46-5.78), diagnosis with acute respiratory tract infection (adjusted OR, 2.62; 95% CI = 1.03-6.69), and urinary tract infection (adjusted OR, 4.69; 95% CI = 2.09-10.56) were associated with higher prescriptions of Watch-group antibiotics. Conclusionsa higher prescribing of Watch-group antibiotics and poor adherence to antibiotic guidelines were observed, especially for children. The findings of this study identified targets for further improvement and interventions needed to develop better antibiotic-prescribing practices.

Factors that may improve paediatric workplace-based assessments: an exploratory study

ObjectivesTo establish if paediatric trainees are satisfied with the current workplace-based assessment (WBA) process. To identify factors that contribute both positively and negatively to the educational experience during WBAs. To...

Perceptual quality assessment of panoramic stitched contents for immersive applications: a prospective survey

The recent advancements in the field of Virtual Reality (VR) and Augmented Reality (AR) have a substantial impact on modern day technology by digitizing each and everything related to human life and open the doors to the next generation Software Technology (Soft Tech). VR and AR technology provide astonishing immersive contents with the help of high quality stitched panoramic contents and 360° imagery that widely used in the education, gaming, entertainment, and production sector. The immersive quality of VR and AR contents are greatly dependent on the perceptual quality of panoramic or 360° images, in fact a minor visual distortion can significantly degrade the overall quality. Thus, to ensure the quality of constructed panoramic contents for VR and AR applications, numerous Stitched Image Quality Assessment (SIQA) methods have been proposed to assess the quality of panoramic contents before using in VR and AR. In this survey, we provide a detailed overview of the SIQA literature and exclusively focus on objective SIQA methods presented till date. For better understanding, the objective SIQA methods are classified into two classes namely Full-Reference SIQA and No-Reference SIQA approaches. Each class is further categorized into traditional and deep learning-based methods and examined their performance for SIQA task. Further, we shortlist the publicly available benchmark SIQA datasets and evaluation metrices used for quality assessment of panoramic contents. In last, we highlight the current challenges in this area based on the existing SIQA methods and suggest future research directions that need to be target for further improvement in SIQA domain.

Acid-Responsive Aggregated Gold Nanoparticles for Radiosensitization and Synergistic Chemoradiotherapy in the Treatment of Esophageal Cancer.

Radiotherapy and chemotherapy are limited by insufficient therapeutic efficacy of low-dose radiation and nonspecific drug biodistribution. Herein, an acid-responsive aggregated nanosystem (AuNPs-D-P-DA) loaded with doxorubicin (DOX) is designed for radiosensitization and synergistic chemoradiotherapy. In response to the acid microenvironment of esophageal cancer (EC), small-sized AuNPs-D-P-DA forms large-sized gold nanoparticle (AuNPs) aggregates in tumor tissues to hinder the backflow of AuNPs to the circulation, resulting in enhanced tumor accumulation and retention. Simultaneously, the AuNPs-based radiosensitization is significantly improved because of the high concentration and large size of intratumoral AuNPs, while DOX are delivered and released specifically into tumor cells triggered by the acid microenvironment for chemo-radio synergistic therapy. Acid-responsive AuNPs exacerbate radiation-induced DNA damage, cell apoptosis, cell cycle arrest, and low colony formation ability in vitro and enhance anti-tumor efficacy in vivo compared to un-responsive control. When combined with acid-responsive DOX, the therapeutic efficacy of the formulation is further improved by their synergistic effect. After the treatment of acid-responsive AuNPs plus radiotherapy, fatty acid metabolism is reprogrammed in xenograft models, which provides potential targets for further improvement of radiosensitization. In summary, the acid-responsive AuNPs-D-P-DA nanosystem leverages the radio- and chemotherapeutic synergies of AuNPs-sensitized X-ray irradiation and acid-responsive DOX in the treatment of EC.

Exploring functionality of the reverse \u03b2-oxidation pathway in Corynebacterium glutamicum for production of adipic acid

BackgroundAdipic acid, a six-carbon platform chemical mainly used in nylon production, can be produced via reverse β-oxidation in microbial systems. The advantages posed by Corynebacterium glutamicum as a model cell factory for implementing the pathway include: (1) availability of genetic tools, (2) excretion of succinate and acetate when the TCA cycle becomes overflown, (3) initiation of biosynthesis with succinyl-CoA and acetyl-CoA, and (4) established succinic acid production. Here, we implemented the reverse β-oxidation pathway in C. glutamicum and assessed its functionality for adipic acid biosynthesis.ResultsTo obtain a non-decarboxylative condensation product of acetyl-CoA and succinyl-CoA, and to subsequently remove CoA from the condensation product, we introduced heterologous 3-oxoadipyl-CoA thiolase and acyl-CoA thioesterase into C. glutamicum. No 3-oxoadipic acid could be detected in the cultivation broth, possibly due to its endogenous catabolism. To successfully biosynthesize and secrete 3-hydroxyadipic acid, 3-hydroxyadipyl-CoA dehydrogenase was introduced. Addition of 2,3-dehydroadipyl-CoA hydratase led to biosynthesis and excretion of trans-2-hexenedioic acid. Finally, trans-2-enoyl-CoA reductase was inserted to yield 37 µg/L of adipic acid.ConclusionsIn the present study, we engineered the reverse β-oxidation pathway in C. glutamicum and assessed its potential for producing adipic acid from glucose as starting material. The presence of adipic acid, albeit small amount, in the cultivation broth indicated that the synthetic genes were expressed and functional. Moreover, 2,3-dehydroadipyl-CoA hydratase and β-ketoadipyl-CoA thiolase were determined as potential target for further improvement of the pathway.

Investigation of PM2.5 pollution during COVID-19 pandemic in Guangzhou, China

The COVID-19 pandemic has raised awareness about various environmental issues, including PM2.5 pollution. Here, PM2.5 pollution during the COVID-19 lockdown was traced and analyzed to clarify the sources and factors influencing PM2.5 in Guangzhou, with an emphasis on heavy pollution. The lockdown led to large reductions in industrial and traffic emissions, which significantly reduced PM2.5 concentrations in Guangzhou. Interestingly, the trend of PM2.5 concentrations was not consistent with traffic and industrial emissions, as minimum concentrations were observed in the fourth period (3/01-3/31, 22.45 μg/m3) of the lockdown. However, the concentrations of other gaseous pollutants, e.g., SO2, NO2 and CO, were correlated with industrial and traffic emissions, and the lowest values were noticed in the second period (1/24-2/03) of the lockdown. Meteorological correlation analysis revealed that the decreased PM2.5 concentrations during COVID-19 can be mainly attributed to decreased industrial and traffic emissions rather than meteorological conditions. When meteorological factors were included in the PM2.5 composition and backward trajectory analyses, we found that long-distance transportation and secondary pollution offset the reduction of primary emissions in the second and third stages of the pandemic. Notably, industrial PM2.5 emissions from western, southern and southeastern Guangzhou play an important role in the formation of heavy pollution events. Our results not only verify the importance of controlling traffic and industrial emissions, but also provide targets for further improvements in PM2.5 pollution.

Metabolic engineering of Pseudomonas putida for production of vanillylamine from lignin-derived substrates.

SummaryWhole‐cell bioconversion of technical lignins using Pseudomonas putida strains overexpressing amine transaminases (ATAs) has the potential to become an eco‐efficient route to produce phenolic amines. Here, a novel cell growth‐based screening method to evaluate the in vivo activity of recombinant ATAs towards vanillylamine in P. putida KT2440 was developed. It allowed the identification of the native enzyme Pp‐SpuC‐II and ATA from Chromobacterium violaceum (Cv‐ATA) as highly active towards vanillylamine in vivo. Overexpression of Pp‐SpuC‐II and Cv‐ATA in the strain GN442ΔPP_2426, previously engineered for reduced vanillin assimilation, resulted in 94‐ and 92‐fold increased specific transaminase activity, respectively. Whole‐cell bioconversion of vanillin yielded 0.70 ± 0.20 mM and 0.92 ± 0.30 mM vanillylamine, for Pp‐SpuC‐II and Cv‐ATA, respectively. Still, amine production was limited by a substantial re‐assimilation of the product and formation of the by‐products vanillic acid and vanillyl alcohol. Concomitant overexpression of Cv‐ATA and alanine dehydrogenase from Bacillus subtilis increased the production of vanillylamine with ammonium as the only nitrogen source and a reduction in the amount of amine product re‐assimilation. Identification and deletion of additional native genes encoding oxidoreductases acting on vanillin are crucial engineering targets for further improvement.

Complications of external cerebrospinal fluid drainage in aneurysmal subarachnoid haemorrhage

BackgroundThe need for external cerebrospinal fluid (CSF) drains in aneurysmal subarachnoid haemorrhage (aSAH) patients is common and might lead to additional complications.ObjectiveA relation between the presence of an external CSF drain and complication risk is investigated.MethodsA prospective complication registry was analysed retrospectively. We included all adult aSAH patients admitted to our academic hospital between January 2016 and January 2018, treated with an external CSF drain. Demographic data, type of external drain used, the severity of the aSAH and complications, up to 30 days after drain placement, were registered. Complications were divided into (1) complications with a direct relation to the external CSF drain and (2) complications that could not be directly related to the use of an external CSF drain referred to as medical complicationsResultsOne hundred and forty drains were implanted in 100 aSAH patients. In total, 112 complications occurred in 59 patients. Thirty-six complications were drain related and 76 were medical complications. The most common complication was infection (n = 34). Drain dislodgement occurred 16 times, followed by meningitis (n = 11) and occlusion (n = 9). A Poisson model showed that the mean number of complications raised by 2.9% for each additional day of drainage (95% CI: 0.6–5.3% p = 0.01).ConclusionComplications are common in patients with aneurysmal subarachnoid haemorrhage of which 32% are drain-related. A correlation is present between drainage period and the number of complications. Therefore, reducing drainage period could be a target for further improvement of care.

Evaluating environmental performance using data envelopment analysis: The case of European countries

Environmental efficiency is considered to be a critical pillar of sustainable development; consequently, improving environmental performance has aroused attention at both the local and global level. This study evaluated the environmental performance of European Union (EU) countries using the data envelopment analysis (DEA) approach and the global Malmquist-Luenberger index. Although literature using the DEA approach to measure environmental performance exists, this study’s main contribution was considering different types of undesirable outputs and using long-term panel data on EU countries. In particular, the DEA window analysis technique was applied to evaluate the environmental performance of 27 EU countries in cross-sectional and time-varying data during the period 2000–2017. Three DEA models were examined; labor, capital, and energy were used as common inputs with a combination of different outputs, including gross domestic product, carbon dioxide and particulate matter emissions, and waste. The empirical results revealed that the trends in the environmental performance of the entire EU and its individual countries were similar under all examined models. Environmental performance was indeed negatively affected by the financial crisis of 2007–2008; this impact was mainly observed in eastern EU countries. Furthermore, both economic and environmental variables significantly influenced countries’ overall efficiency. The analysis using the global Malmquist-Luenberger index elucidated that overall, EU countries experienced the efficiency improvement during the study period, although fluctuations were observed. These results enable countries and policymakers to understand their environmental performance, identify strengths and weaknesses, and set targets for further improvement based on the current best practices of comparable peers.

Mental illness stigma after a decade of Time to Change England: inequalities as targets for further improvement.

BackgroundEngland’s Time to Change programme to reduce mental health-related stigma and discrimination included a social marketing campaign using traditional and social media, and targeted middle-income groups aged 25–45 between 2009 and 2016. From 2017, the same age group on low to middle incomes were targeted, and the content focused on men’s mental health, by changing the advertising and adapting the ‘key messages’. This study investigates changes in stigma-related public knowledge, attitudes and desire for social distance in England since Time to Change began in 2008–19 and for 2017–19.MethodsUsing data from a face-to-face survey of a nationally representative quota sample of adults for England, we evaluated longitudinal trends in outcomes with regression analyses and made assumptions based on a simple random sample. The pre-existing survey used a measure of attitudes; measures of knowledge and desire for social distance were added in 2009.ResultsReported in standard deviation units (95% CI), the improvement for knowledge for 2009–19 was 0.25 (0.19, 0.32); for attitudes, 2008–19, 0.32 (0.26, 0.39) and for desire for social distance, 2009–19 0.29 (0.23, 0.36). Significant interactions between year and both region and age suggest greater improvements in London, where stigma is higher, and narrowing of age differences. There were significant improvements between 2017 and 2019 in knowledge [0.09 (0.02, 0.16)] and attitudes [0.08 (0.02, 0.14)] but not social distance.ConclusionThe positive changes support the effectiveness of Time to Change but cannot be definitively attributed to it. Inequalities in stigma by demographic characteristics present targets for research and intervention.

Genetics and Breeding of Lupinus mutabilis: An Emerging Protein Crop.

Protein crops have gained increasing interest in recent years, as a transition towards plant-protein based diets appears pivotal to ensure global food security and preserve the environment. The Andean species Lupinus mutabilis emerges as an ideal protein crop with great potential for Europe and other regions with temperate climates. This species is characterized by oil and protein content similar to soybean and is highly valued for its adaptability to colder climates and low input agriculture on marginal land. However, its introduction outside the Andes has yet to take off. To date, L. mutabilis remains an under-studied crop, lacking high yield, early maturity and a consistent breeding history. This review paper identifies L. mutabilis limitations and potential uses, and suggests the main breeding targets for further improvement of this crop. It also highlights the potential of new molecular tools and available germplasm resources that can now be used to establish L. mutabilis as a viable protein crop.

Assessing the impact of geographical access to health facilities on maternal healthcare utilization: evidence from the Burkina Faso demographic and health survey 2010

BackgroundImproving maternal and child health (MCH) remains a serious challenge for many developing countries. Geographical accessibility from a residence to the nearest health facility is suspected to be an important obstacle hampering the use of appropriate services for MCH especially in Sub-Sharan African countries. In Burkina Faso, a landlocked country in the Sahel region of West Africa, women’s use of proper healthcare services during pregnancy and childbirth is still low. This study therefore assessed the impact of geographical access to health facilities on maternal healthcare utilization in Burkina Faso.MethodsWe used the Burkina Faso demographic and health survey (DHS) 2010 dataset, with its sample of 10,364 mothers aged 15–49 years. Distance from residential areas to the closest health facility was measured by merging the DHS dataset with Geographic Information System data on the location of health centers in Burkina Faso. Multivariate logistic regressions were conducted to estimate the effects of distance on maternal healthcare utilization.ResultsRegression results revealed that the longer the distance to the closest health center, the less likely it is that a woman will receive appropriate maternal healthcare services. The estimates show that one kilometer increase in distance to the closest health center reduces the odds that a woman will receive four or more antenatal care by 0.05 and reduces by 0.267 the odds that she will deliver her baby with the assistance of a skilled birth attendant.ConclusionsImproving geographical access to health facilities increases the use of appropriate healthcare services during pregnancy and childbirth. Investment in transport infrastructure should be a prioritized target for further improvement in MCH in Burkina Faso.

Acute and midterm outcomes of the post-approval MELODY Registry: a multicentre registry of transcatheter pulmonary valve implantation.

The post-approval MELODY Registry aimed to obtain multicentre registry data after transcatheter pulmonary valve implantation (TPVI) with the Melody™ valve (Medtronic plc.) in a large-scale cohort of patients with congenital heart disease (CHD). Retrospective analysis of multicentre registry data after TPVI with the Melody™ valve. Eight hundred and forty-five patients (mean age: 21.0 ± 11.1 years) underwent TPVI in 42 centres between December 2006 and September 2013 and were followed-up for a median of 5.9 years (range: 0-11.0 years). The composite endpoint of TPVI-related events during follow-up (i.e. death, reoperation, or reintervention >48 h after TPVI) showed an incidence rate of 4.2% per person per year [95% confidence interval (CI) 3.7-4.9]. Transcatheter pulmonary valve implantation infective endocarditis (I.E.) showed an incidence rate of 2.3% per person per year (95% CI 1.9-2.8) and resulted in significant morbidity and in nine deaths. In multivariable Cox proportional hazard models, the invasively measured residual right ventricle (RV)-to-pulmonary artery (PA) pressure gradient (per 5 mmHg) was associated with the risk of the composite endpoint (adjusted hazard ratio: 1.21, 95% CI 1.12-1.30; P < 0.0001) and the risk of TPVI I.E. (adjusted hazard ratio: 1.19, 95% CI 1.07-1.32; P = 0.002). Major procedural complications (death, surgical, or interventional treatment requirement) occurred in 0.5%, 1.2%, and 2.0%, respectively. Acutely, the RV-to-PA pressure gradient and the percentage of patients with pulmonary regurgitation grade >2 improved significantly from 36 [interquartile range (IQR) 24-47] to 12 (IQR 7-17) mmHg and 47 to 1%, respectively (P < 0.001 for each). The post-approval MELODY Registry confirms the efficacy of TPVI with the Melody™ valve in a large-scale cohort of CHD patients. The residual invasively measured RV-to-PA pressure gradient may serve as a target for further improvement in the composite endpoint and TPVI I.E. However, TPVI I.E. remains a significant concern causing significant morbidity and mortality.

Ten-year follow up of patients with first-episode schizophrenia spectrum disorder from an early intervention service: Predictors of clinical remission and functional recovery

The long-term recovery rate of patients with schizophrenia-spectrum disorders has been persistently low despite the implementation of early intervention (EI) services internationally. It is, therefore, important to identify the modifiable factors during the early stage of the illness that predict long-term remission and recovery. The aim of this study is to explore the predictive value of the early stage clinical factors on the clinical remission and functional recovery at 10-year follow-up of patients with schizophrenia-spectrum disorders who received a 2-year EI service. Patients who received the EI service throughout the region of Hong Kong between 1st July 2001 and 30th June 2002 and with a diagnosis of schizophrenia-spectrum disorder were identified from the centralized hospital database (Clinical Management system, CMS). Semi-structured clinical interview was conducted at 10-year follow-up with a successful interview rate of 74.3% (n = 107). Clinical data was systematically retrieved each month for the first three years from the CMS and written clinical records using a standardized data entry form based on operationalized definitions. Results found shorter duration of untreated psychosis (DUP) predicted long-term clinical remission; higher educational level and shorter period of unemployment during the initial three years of the illness predicted functional recovery. Higher educational level, longer period of employment and planned medication discontinuation during the initial three years predicted complete recovery. The current study demonstrates the long-term impact of DUP and suggests improvement of employment during the early stage of illness could be a potential target for further improvement of the service.

RNAi assisted genome evolution unveils yeast mutants with improved xylose utilization.

Xylose is a major component of lignocellulosic biomass, one of the most abundant feedstocks for biofuel production. Therefore, efficient and rapid conversion of xylose to ethanol is crucial in the viability of lignocellulosic biofuel plants. In this study, RNAi Assisted Genome Evolution (RAGE) was used to improve the xylose utilization rate in SR8, one of the most efficient publicly available xylose utilizing Saccharomyces cerevisiae strains. To identify gene targets for further improvement, we created a genome-scale library consisting of both genetic over-expression and down-regulation mutations in SR8. Followed by screening in media containing xylose as the sole carbon source, yeast mutants with 29% faster xylose utilization, and 45% higher ethanol productivity were obtained relative to the parent strain. Two known and two new effector genes were identified in these mutant strains. Notably, down-regulation of CDC11, an essential gene, resulted in faster xylose utilization, and this gene target cannot be identified in genetic knock-out screens.

Changes in structure and function of bacterial communities during coconut leaf vermicomposting.

To understand bacterial community dynamics during the vermicomposting of lignin-rich coconut leaves using an indigenous isolate of an epigeic earthworm, Eudrilus sp., we employed amplicon-based pyrosequencing of the V1 to V3 region of the 16S rRNA genes. Total community DNA was isolated from two separate vermicomposting tanks in triplicate at four different stages of the process: pre-decomposition (15th day), initial vermicomposting (45th day), 50-70% vermicomposting (75th day) and mature vermicompost (105th day). Alpha diversity measurements revealed an increase in bacterial diversity till the 75th day, which then declined in the mature vermicompost. Beta diversity comparisons showed formation of distinct, stage-specific communities. In terms of relative abundance, the Acidobacteria, Actinobacteria, Chloroflexi, Gemmatimonadetes, Nitrospirae, Planctomycetes, TM7 and WS3 groups increased until the 50-70% vermicomposting stage (p=0.05). During the same time, the abundance of Bacteroidetes and Proteobacteria decreased. In contrast, the levels of Firmicutes increased throughout the 105-day vermicomposting process. The distribution of the most abundant OTUs revealed that each stage of the vermicomposting process possessed its own unique microbiome. Predictions based on the OTUs present by PICRUSt suggested a functional shift in the microbiome during vermicomposting. Enzymes and pathways of lipid and lignin metabolism were predicted to be initially abundant, but by the end of the process, biosynthesis of secondary metabolites and plant beneficial properties were enriched. The study revealed that bacterial communities undergo a continuous change throughout the vermicomposting process and that certain OTUs associated with specific stages could be targets for further improvements in the process.

Rational design and metabolic analysis of Escherichia coli for effective production of L-tryptophan at high concentration.

L-tryptophan (L-trp) is a biosynthetic precursor of various bioactive components with pharmaceutical interest. The development of an efficient L-trp production strain using targeted molecular engineering approaches is challenging due to the requirement of several precursors and the complex regulations of the pathways involved. In this study, we present a rationally engineered and genetically stable L-trp overproducing Escherichia coli strain. The streamlined strain E. coli S028 is able to efficiently produce 34-40g/L of L-trp with a yield of 0.15gL-trp/g glucose and a productivity of 0.60g/L/h in fed-batch fermentations. The titer and productivity of L-trp achieved are over twice as much as those reported so far for rationally developed L-trp producers. In addition, for the first time, both intracellular and extracellular concentrations of L-trp and the key metabolites in a L-trp hyperproducer strain were measured with an automated fast-sampling unit which is connected to a well-controlled bioreactor. The time series metabolic analysis gives valuable information about the regulation of L-trp synthesis in a highly productive strain and reveals targets for further improvement. Among others, it was found that L-trp and the byproduct glutamate (L-glu) accumulated to an extremely high level in the cell initially whereas the intracellular concentrations of glutamine (L-gln) stayed at a relatively low level throughout the fermentation. The metabolic analysis suggests that (a) the engineered serine biosynthesis pathway was able to effectively synthesize the substrate serine (intracellular concentration>8mM) for L-trp production, while (b) the substrate L-gln with an intracellular concentration of 0.8-1.2mM seems to limit the biosynthesis of L-trp, even though L-glu was overproduced intra- and extracellularly. Thus, an increased availability of glutamine synthetase which catalyzes L-glu conversion to L-gln and an overexpression of the L-trp exporter gene could be important targets for further strain improvement.

Elucidation of ethanol tolerance mechanisms in Saccharomyces cerevisiae by global metabolite profiling.

Ethanol, the major fermentation product of yeast, is a stress factor in yeast. We previously constructed an ethanol-tolerant mutant yeast iETS3 by using the global transcriptional machinery engineering. However, the ethanol-tolerance mechanism has not been systematically investigated. In this study, global metabolite profiling was carried out, mainly by gas chromatography/time-of-flight mass spectrometry (GC/TOF MS), to investigate the mechanisms of ethanol tolerance in iETS3. A total of 108 intracellular metabolites were identified by GC/TOF MS and high performance liquid chromatography, and these metabolites were mostly intermediates of the central carbon metabolism. The metabolite profiles of iETS3 and BY4741, cultured with or without ethanol, were significantly different based on principal component and hierarchical clustering analyses. Our metabolomic analyses identified the compositional changes in cell membranes and the activation of glutamate metabolism and the trehalose synthetic pathway as the possible mechanisms for the ethanol tolerance. These metabolic traits can be considered possible targets for further improvement of ethanol tolerance in the mutant. For example, the KGD1 deletion mutant, with up-regulated glutamate metabolism, showed increased tolerance to ethanol. This study has demonstrated that metabolomics can be a useful tool for strain improvement and phenotypic analysis of microorganisms under stress.