2494 research outputs found
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Enzymatic process optimization for the in vitro production of isoprene from mevalonate
No full textAbstract
Background
As an important bulk chemical for synthetic rubber, isoprene can be biosynthesized by robust microbes. But rational engineering and optimization are often demanded to make the in vivo process feasible due to the complexities of cellular metabolism. Alternative synthetic biochemistry strategies are in fast development to produce isoprene or isoprenoids in vitro.
Results
This study set up an in vitro enzyme synthetic chemistry process using 5 enzymes in the lower mevalonate pathway to produce isoprene from mevalonate. We found the level and ratio of individual enzymes would significantly affect the efficiency of the whole system. The optimized process using 10 balanced enzyme unites (5.0\ua0\ub5M of MVK, PMK, MVD; 10.0\ua0\ub5M of IDI, 80.0\ua0\ub5M of ISPS) could produce 6323.5\ua0\ub5mol/L/h (430\ua0mg/L/h) isoprene in a 2\ua0ml in vitro system. In a scale up process (50\ua0ml) only using 1 balanced enzyme unit (0.5\ua0\ub5M of MVK, PMK, MVD; 1.0\ua0\ub5M of IDI, 8.0\ua0\ub5M of ISPS), the system could produce 302\ua0mg/L isoprene in 40\ua0h, which showed higher production rate and longer reaction phase with comparison of the in vivo control.
Conclusions
By optimizing the enzyme levels of lower MVA pathway, synthetic biochemistry methods could be set up for the enzymatic production of isoprene or isoprenoids from mevalonate
Efficient method for establishing F1 progeny from wild populations of Anopheles mosquitoes
No full textAbstract
Background
The changing malaria situation in Madagascar requires additional knowledge on the physiology and behaviour of local mosquito vectors. However, the absence of established colonies for several anopheline species present in Madagascar constitutes a limiting factor. To avoid labour intensive work and uncertainty for success of establishing Anopheles colonies from Malagasy species, field collections of blood-fed females and in-tube forced oviposition were combined to reliably produce large numbers of F1 progeny.
Methods
Blood-fed females were captured in zebu stables or open zebu parks. Oviposition was induced by enclosing gravid females in eppendorf tubes as initially described for Anopheles funestus . The effect of cold anaesthesia on inducing in-tube forced oviposition and on egg yield was assessed for five Anopheles species, namely Anopheles coustani , An. funestus , Anopheles mascarensis , Anopheles arabiensis and Anopheles squamosus . The production of eggs from in-tube forced oviposition and standard egg laying in cages was compared.
Results
For the five anopheline species studied, the in-tube forced oviposition method had different efficacy ranging from 35.6 to 71.1% females willing to lay eggs in tubes. Interestingly, prior anaesthesia increased significantly the proportion of ovipositing females for An. mascarensis . Prior anaesthesia has a marginal effect on the number of eggs produced. However, the overall yield in eggs collected using the in-tube forced oviposition method largely exceeds the number of eggs that can be produced by females free to oviposit in cages.
Conclusion
The efficiency of the method allowed the production of F1 progeny in numbers sufficiently large for developing detailed analyses of the five species tested, including behavioural studies, insecticide resistance assessment and molecular characterization, as well as vector competence studies. It should be applicable to other anopheline species difficult to colonize
Gender bias and sex-based differences in health care efficiency in Polish regions
No full textAbstract
Background
Health differences between sexes are relatively well recognized, though less is known about the specificity of women's and men's health responsiveness to medical care. Applying data from Polish regions, this study identifies sex-based differences in medical care efficiency and investigates the reasons for these disparities in the gender bias context.
Methods
The study estimates sex-specific health production functions for regional data from Poland (1999\u20132013). Using panel-data regression, male and female life expectancies at ages 0, 15, 30, 45, 60 and 65 are regressed on a set of socioeconomic factors, with the primary interest in medical care proxied by doctor density.
Results
The results show that in Poland the association between life expectancy and doctor density was positive for both men and women; however, the coefficients for medical care were insignificant for those at birth and at the age of 30 for both sexes. The magnitude of health care for longevity was higher for men comparing to women at every age, though the difference between sexes was not statistically significant. The sex-based disparities in medical care efficiency were more pronounced at younger ages and they diminished with age. The inspection of data on the health system in Poland shows that male patients seemed to be in an advantageous position: the mean reimbursement per service for men was higher in most medical care areas; men reported less problems with access to health care; and their mortality trend exhibited more favorable evolution over time. Additionally, the association between other socioeconomic factors and health also differed across sexes, and several of these factors were more important for life expectancy than health care.
Conclusion
Polish medical care suffers from gender bias, which possibly makes men more responsive to medical care. The disparities in the operation of medical care in Poland should be challenged to achieve more equal access to services between sexes and possibly to gain more health from the treatment of female patients
HECTD1 controls the protein level of IQGAP1 to regulate the dynamics of adhesive structures
No full textAbstract
Background
Cell migration including collective cell movement and individual cell migration are crucial factors in embryogenesis. During the spreading/migration of cells, several types of adhesive structures physically interacting with the extracellular matrix (ECM) or with another cell have been described and the formation and maturation of adhesion structures are coordinated, however the molecular pathways involved are still not fully understood.
Results
We generated a mouse embryonic fibroblast line (MEF) from homozygous mutant ( Hectd1 R/R
, Hectd1 Gt(RRC200)
) mouse of the E3 ubiquitin ligase for inhibin B receptor ( Hectd1 ). Detailed examination of cell motion on MEF cells demonstrated that loss of Hectd1 resulted in accelerated cell spreading and migration but impaired directionality of migration. In Hectd1 R/R
cells paxillin and zyxin were largely mis-localized, whereas their expression levels were unchanged. In addition the formation of focal adhesions (FAs) was impaired and the focal complexes (FXs) were increased. We further identified HECTD1 as a key regulator of IQGAP1. IQGAP1 co-localized together with HECTD1 in the leading edge of cells. HECTD1 interacted with IQGAP1 and regulated its degradation through ubiquitination. Over-expression of IQGAP1 in control MEF phenocopied the spreading and migration defects of Hectd1 R/R
cells. In contrast, siRNA-mediated knockdown of IQGAP1 rescued the defects in cellular movement of Hectd1 R/R
cells.
Conclusions
The E3 ligase activity of Hectd1 regulates the protein level of IQGAP1 through ubiquitination and therefore mediates the dynamics of FXs including the recruitment of paxillin and actinin. IQGAP1 is one of the effectors of HECTD1
The predictive capabilities of a novel cardiovascular magnetic resonance derived marker of cardiopulmonary reserve on established prognostic surrogate markers in patients with pulmonary vascular disease: results of a longitudinal pilot study
No full textAbstract
Background
No unified method exists to effectively predict and monitor progression of pulmonary arterial hypertension (PAH). We assessed the longitudinal relationship between a novel marker of cardiopulmonary reserve and established prognostic surrogate markers in patients with pulmonary vascular disease.
Methods and Results
Twenty participants with confirmed ( n \u2009=\u200914) or at high risk ( n \u2009=\u20096) for PAH underwent cardiovascular magnetic resonance (CMR) at baseline and after ~6\ua0months of guideline-appropriate management. Ten PAH participants underwent RHC within 48\ua0h of each CMR. RHC (mean pulmonary arterial pressure, mPAP; pulmonary vascular resistance index, PVRI; cardiac index, CI) and phase-contrast CMR (mean pulmonary arterial blood flow velocity, meanPAvel) measurements were taken at rest and during continuous adenosine infusion (70/140/210 mcg/kg/min). Initial meanPAvel\u2019s (rest and hyperemic) were correlated with validated surrogate prognostic parameters (CMR: RV ejection fraction, RVEF; RV end systolic volume indexed, RVESVI; RHC: PVRI, CI; biomarker: NT-pro brain natriuretic peptide, NTpBNP; clinical: 6-min walk distance, 6MWD), a measure of pulmonary arterial stiffness (elastic modulus) and volumetric estimation of RV ventriculoarterial (VA) coupling. Changes in meanPAvel\u2019s were correlated with changes in comparator parameters over time.
At initial assessment, meanPAvel at rest correlated significantly with PVRI (inversely), CI (positively) and elastic modulus (inversely) ( R
2
\u2009>\u20090.37, P \u2009<\u20090.05 for all), whereas meanPAvel at peak hyperemia correlated significantly with PVRI, RVEF, RVESVI, 6MWD, elastic modulus and VA coupling ( R
2
\u2009>\u20090.30, P \u2009<\u20090.05 for all). Neither resting or hyperemia-derived meanPAvel correlated with NTpBNP levels. Initial meanPAvel at rest correlated significantly with RVEF, RVESVI, CI and VA coupling at follow up assessment ( R
2
\u2009>\u20090.2, P \u2009<\u20090.05 for all) and initial meanPAvel at peak hyperemia correlated with RVEF, RVESVI, PVRI and VA coupling ( R
2
\u2009>\u20090.37, P ..
Systematic epistatic mapping of cellular processes
No full textAbstract
Genetic screens have identified many novel components of various biological processes, such as components required for cell cycle and cell division. While forward genetic screens typically generate unstructured \u2018hit\u2019 lists, genetic interaction mapping approaches can identify functional relations in a systematic fashion. Here, we discuss a recent study by our group demonstrating a two-step approach to first screen for regulators of the mitotic cell cycle, and subsequently guide hypothesis generation by using genetic interaction analysis. The screen used a high-content microscopy assay and automated image analysis to capture defects during mitotic progression and cytokinesis. Genetic interaction networks derived from process-specific features generate a snapshot of functional gene relations in those processes, which follow a temporal order during the cell cycle. This complements a recently published approach, which inferred directional genetic interactions reconstructing hierarchical relationships between genes across different phases during mitotic progression. In conclusion, this strategy leverages unbiased, genome-wide, yet highly sensitive and process-focused functional screening in cells
FOXN1 deficient nude severe combined immunodeficiency
No full textAbstract
Nude severe combined immunodeficiency is a rare inherited disease caused by autosomal recessive loss-of-function mutations in FOXN1 . This gene encodes a transcription factor essential for the development of the thymus, the primary lymphoid organ that supports T-cell development and selection. To date nine cases have been reported presenting with the clinical triad of absent thymus resulting in severe T-cell immunodeficiency, congenital alopecia universalis and nail dystrophy. Diagnosis relies on testing for FOXN1 mutations, which allows genetic counselling and guides therapeutic management. Options for treating the underlying immune deficiency include HLA-matched genoidentical haematopoietic cell transplantation containing mature donor T-cells or thymus tissue transplantation. Experience from other severe combined immune deficiency syndromes suggests that early diagnosis, supportive care and definitive management result in better patient outcomes. Without these the prognosis is poor due to early-onset life threatening infections
Integrated omics analyses reveal the details of metabolic adaptation of Clostridium thermocellum to lignocellulose-derived growth inhibitors released during the deconstruction of switchgrass
No full textAbstract
Background
Clostridium thermocellum is capable of solubilizing and converting lignocellulosic biomass into ethanol. Although much of the work-to-date has centered on characterizing this microbe\u2019s growth on model cellulosic substrates, such as cellobiose, Avicel, or filter paper, it is vitally important to understand its metabolism on more complex, lignocellulosic substrates to identify relevant industrial bottlenecks that could undermine efficient biofuel production. To this end, we have examined a time course progression of C. thermocellum grown on switchgrass to assess the metabolic and protein changes that occur during the conversion of plant biomass to ethanol.
Results
The most striking feature of the metabolome was the observed accumulation of long-chain, branched fatty acids over time, implying an adaptive restructuring of C. thermocellum\u2019s cellular membrane as the culture progresses. This is undoubtedly a response to the gradual accumulation of lignocellulose-derived inhibitory compounds as the organism deconstructs the switchgrass to access the embedded cellulose. Corroborating the metabolomics data, proteomic analysis revealed a corresponding time-dependent increase in various enzymes, including those involved in the interconversion of branched amino acids valine, leucine, and isoleucine to iso- and anteiso-fatty acid precursors. Additionally, the metabolic accumulation of hemicellulose-derived sugars and sugar alcohols concomitant with increased abundance of enzymes involved in C5 sugar metabolism/pentose phosphate pathway indicates that C. thermocellum shifts glycolytic intermediates to alternate pathways to modulate overall carbon flux in response to C5 sugar metabolites that increase during lignocellulose deconstruction.
Conclusions
Integrated omic platforms provided complementary systems biological information that highlight C. thermocellum \u2019s specific response to cytotoxic inhibitors released during the deconstruction and utilization of switchgrass. These additional viewpoints allowed us to fully realize the level to which the organism adapts to an increasingly challenging culture environment\u2014information that will prove critical to C. thermocellum ..
Molecular detection of Rickettsia species in ticks collected from the southwestern provinces of the Republic of Korea
No full textAbstract
Background
Rickettsiae constitute a group of arthropod-borne, Gram-negative, obligate intracellular bacteria that are the causative agents of diseases ranging from mild to life threatening that impact on medical and veterinary health worldwide.
Methods
A total of 6,484 ticks were collected by tick drag from June-October 2013 in the southwestern provinces of the Republic of Korea (ROK) (Jeollanam, n \u2009=\u20093,995; Jeollabuk, n \u2009=\u2009680; Chungcheongnam, n \u2009=\u20091,478; and Chungcheongbuk, n \u2009=\u2009331). Ticks were sorted into 311 pools according to species, collection site, and stage of development. DNA preparations of tick pools were assayed for rickettsiae by 17\ua0kDa antigen gene and ompA nested PCR (nPCR) assays and the resulting amplicons sequenced to determine the identity and prevalence of spotted fever group rickettsiae (SFGR).
Results
Haemaphysalis longicornis (4,471; 52 adults, 123 nymphs and 4,296 larvae) were the most commonly collected ticks, followed by Haemaphysalis flava (1,582; 28 adults, 263 nymphs and 1,291 larvae), and Ixodes nipponensis (431; 25 adults, 5 nymphs and 401 larvae). The minimum field infection rate/100 ticks (assuming 1 positive tick/pool) was 0.93% for the 17\ua0kDa antigen gene and 0.82% for the ompA nPCR assays. The partial 17\ua0kDa antigen and ompA gene sequences from positive pools of H. longicornis were similar to: Rickettsia sp. HI550 (99.4\u2013100%), Rickettsia sp. FUJ98 (99.3\u2013100%), Rickettsia sp. HIR/D91 (99.3\u2013100%), and R. japonica (99.7%). One sequence of the partial 17\ua0kDa antigen gene for H. flava was similar to Rickettsia sp. 17kd-005 (99.7%), while seven sequences of the 17\ua0kDa antigen gene obtained from I. nipponensis ticks were similar to R. monacensis IrR/Munich (98.7\u2013100%) and Rickettsia sp. IRS3 (98.9%).
Conclusions
SFG rickettsiae were detected in three species of ixodid ticks collected in the southwestern provinces of the ROK during 2013. A number of rickettsiae have been recently reported from ticks in Korea, some of which were identified as medically important. Results from this study and previous reports demonstrate the need to conduct longitudinal investigations to identify tick-borne rickettsiae and better understand their geographical distributions and potential impact on medical and veterinary health, in addition to risk communication and ..
MS_HistoneDB, a manually curated resource for proteomic analysis of human and mouse histones
No full textAbstract
Background
Histones and histone variants are essential components of the nuclear chromatin. While mass spectrometry has opened a large window to their characterization and functional studies, their identification from proteomic data remains challenging. Indeed, the current interpretation of mass spectrometry data relies on public databases which are either not exhaustive (Swiss-Prot) or contain many redundant entries (UniProtKB or NCBI). Currently, no protein database is ideally suited for the analysis of histones and the complex array of mammalian histone variants.
Results
We propose two proteomics-oriented manually curated databases for mouse and human histone variants. We manually curated >1700 gene, transcript and protein entries to produce a non-redundant list of 83 mouse and 85 human histones. These entries were annotated in accordance with the current nomenclature and unified with the \u201cHistoneDB2.0 with Variants\u201d database. This resource is provided in a format that can be directly read by programs used for mass spectrometry data interpretation. In addition, it was used to interpret mass spectrometry data acquired on histones extracted from mouse testis. Several histone variants, which had so far only been inferred by homology or detected at the RNA level, were detected by mass spectrometry, confirming the existence of their protein form.
Conclusions
Mouse and human histone entries were collected from different databases and subsequently curated to produce a non-redundant protein-centric resource, MS_HistoneDB. It is dedicated to the proteomic study of histones in mouse and human and will hopefully facilitate the identification and functional study of histone variants