Flux Design: In silico design of cell factories based on correlation of pathway fluxes to desired properties

<p>Abstract</p> <p>Background</p> <p>The identification of genetic target genes is a key step for rational engineering of production strains towards bio-based chemicals, fuels or therapeutics. This is often a difficult task, because superior production performance typically requires a combination of multiple targets, whereby the complex metabolic networks complicate straightforward identification. Recent attempts towards target prediction mainly focus on the prediction of gene deletion targets and therefore can cover only a part of genetic modifications proven valuable in metabolic engineering. Efficient in silico methods for simultaneous genome-scale identification of targets to be amplified or deleted are still lacking.</p> <p>Results</p> <p>Here we propose the identification of targets via flux correlation to a chosen objective flux as approach towards improved biotechnological production strains with optimally designed fluxes. The approach, we name Flux Design, computes elementary modes and, by search through the modes, identifies targets to be amplified (positive correlation) or down-regulated (negative correlation). Supported by statistical evaluation, a target potential is attributed to the identified reactions in a quantitative manner. Based on systems-wide models of the industrial microorganisms <it>Corynebacterium glutamicum </it>and <it>Aspergillus niger</it>, up to more than 20,000 modes were obtained for each case, differing strongly in production performance and intracellular fluxes. For lysine production in <it>C. glutamicum </it>the identified targets nicely matched with reported successful metabolic engineering strategies. In addition, simulations revealed insights, e.g. into the flexibility of energy metabolism. For enzyme production in <it>A.niger </it>flux correlation analysis suggested a number of targets, including non-obvious ones. Hereby, the relevance of most targets depended on the metabolic state of the cell and also on the carbon source.</p> <p>Conclusions</p> <p>Objective flux correlation analysis provided a detailed insight into the metabolic networks of industrially relevant prokaryotic and eukaryotic microorganisms. It was shown that capacity, pathway usage, and relevant genetic targets for optimal production partly depend on the network structure and the metabolic state of the cell which should be considered in future metabolic engineering strategies. The presented strategy can be generally used to identify priority sorted amplification and deletion targets for metabolic engineering purposes under various conditions and thus displays a useful strategy to be incorporated into efficient strain and bioprocess optimization.</p

Implementation of seven echocardiographic parameters of myocardial asynchrony to improve the long-term response rate of cardiac resynchronization therapy (CRT)

<p>Abstract</p> <p>Background</p> <p>Cardiac resynchronization Therapy (CRT) is an effective therapy for chronic heart failure with beneficial hemodynamic effects leading to a reduction of morbidity and mortality. The responder rates, however, are low. There are various and contentious echocardiographic parameters of myocardial asynchrony. Patient selection by echocardiographic assessment of asynchrony is thought to improve responder rates.</p> <p>Methods</p> <p>In this small single-center pilot-study, seven established parameters of myocardial asynchrony were used to select patients for CRT: (1) interventricular electromechanical delay (IMD, cut-off ≥ 40 ms), (2) Septal-to-posterior wall motion delay (SPWMD, ≥ 130 ms), (3) maximal difference in time-to-peak velocities between any two of twelve LV segments (Ts-12 ≥ 104 ms), (4) standard deviation of time to peak myocardial velocities (Ts-12-SD, ≥ 34.4 ms), (5) difference between the septal and basal time-to-peak velocity (TDId, ≥ 60 ms), (6) left ventricular electromechanical delay (LVEMD, > 140 ms) and (7) delayed longitudinal contraction (DLC, > 2 segments).</p> <p>16 chronic heart failure patients (NYHA III–IV, LVEF < 0.35, QRS ≥ 120 ms) at least two out of seven parameters of myocardial asynchrony received cardiac resynchronization therapy (CRT-ICD). Follow-up echo examination was after 6 months. The control group was a historic group of CRT patients (n = 38) who had not been screened for echocardiographic signs of myocardial asynchrony prior to device implantation.</p> <p>Results</p> <p>Based on reverse remodeling (relative reduction of LVESV > 15%, relative increase of LVEF > 25%), the responder rate to CRT was 81.2% in patients selected for CRT according to our protocol as compared to 47.4% in the control group (p = 0.04). At baseline, there were on average 4.1 ± 1.6 positive parameters of asynchrony (follow-up: 3.7 [± 1.6] parameters positive, p = 0.52). Only the LVEMD decreased significantly after CRT (p = 0.027). The remaining parameters showed a non-significant trend towards reduction of myocardial asynchrony.</p> <p>Conclusion</p> <p>The implementation of different markers of asynchrony in the selection process for CRT improves the hemodynamic response rate to CRT.</p

Outcome after surgery for prosthetic valve endocarditis and the impact of preoperative treatment

ObjectivesThis study examined the outcomes of surgery for active prosthetic valve endocarditis in a recent decade, with special interest in preoperative treatment and predictors for early and late events.MethodsFrom 2000 to 2010, a cohort of 149 consecutive patients (mean age, 64 ± 13.9 years; 72% were male) underwent redo-surgery for prosthetic valve endocarditis and were reviewed regarding early (≤60 days) and late (>60 days) events (death, reinfection, reoperation). Kaplan–Meier survival curves and Cox regression analysis were used to investigate the impact of preoperative intervals and predictors for events, respectively.ResultsPreoperative status was critical (European System for Cardiac Operative Risk Evaluation >20%) in 121 patients (81.2%). Staphylococci were the most common infecting microorganisms (27.5%). The median interval between onset of symptoms and diagnosis and between diagnosis and operation was 2 days (interquartile range, 1-5) and 8 days (interquartile range, 2-23), respectively. Operative mortality (≤30 days) was 12.8%. Mean follow-up was 4 ± 2.9 years. In 53 patients, 47 early (24 deaths, 14 recurrences, 9 reoperations) and 22 late events (11 deaths, 9 recurrences, 2 reoperations) occurred. Overall and event-free survivals at 10 years were 75% ± 3.8% and 64% ± 4.0%, respectively. Freedom from recurrent infection and reoperation at 10 years were 81% ± 3.6% and 91% ± 2.6%, respectively. In multivariate Cox regression, mechanical circulatory support, prolongation between onset of symptoms and diagnosis more than 30 days, and preoperative presence of renal failure predicted early events, and double valve replacement predicted late events.ConclusionsCardiac and renal function, need for double valve replacement, and preoperative treatment predicted outcomes. A prolonged interval in which patients were left untreated while symptomatic, but not prolongation of preoperative antibiotic treatment, increased risk

Comprehensive Identification of Immunodominant Proteins of Brucella abortus and Brucella melitensis Using Antibodies in the Sera from Naturally Infected Hosts

Brucellosis is a debilitating zoonotic disease that affects humans and animals. The diagnosis of brucellosis is challenging, as accurate species level identification is not possible with any of the currently available serology-based diagnostic methods. The present study aimed at identifying Brucella (B.) species-specific proteins from the closely related species B. abortus and B. melitensis using sera collected from naturally infected host species. Unlike earlier reported investigations with either laboratory-grown species or vaccine strains, in the present study, field strains were utilized for analysis. The label-free quantitative proteomic analysis of the naturally isolated strains of these two closely related species revealed 402 differentially expressed proteins, among which 63 and 103 proteins were found exclusively in the whole cell extracts of B. abortus and B. melitensis field strains, respectively. The sera from four different naturally infected host species, i.e., cattle, buffalo, sheep, and goat were applied to identify the immune-binding protein spots present in the whole protein extracts from the isolated B. abortus and B. melitensis field strains and resolved on two- dimensional gel electrophoresis. Comprehensive analysis revealed that 25 proteins of B. abortus and 20 proteins of B. melitensis were distinctly immunoreactive. Dihydrodipicolinate synthase, glyceraldehyde-3-phosphate dehydrogenase and lactate/malate dehydrogenase from B. abortus, amino acid ABC transporter substrate-binding protein from B. melitensis and fumarylacetoacetate hydrolase from both species were reactive with the sera of all the tested naturally infected host species. The identified proteins could be used for the design of serological assays capable of detecting pan- Brucella, B. abortus- and B. melitensis-specific antibodies

Synthesis and transfer of galactolipids in the chloroplast envelope membranes of \u3ci\u3eArabidopsis thaliana\u3c/i\u3e

Galactolipids [monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG)] are the hallmark lipids of photosynthetic membranes. The galactolipid synthases MGD1 and DGD1 catalyze consecutive galactosyltransfer reactions but localize to the inner and outer chloroplast envelopes, respectively, necessitating intermembrane lipid transfer. Here we show that the N-terminal sequence of DGD1 (NDGD1) is required for galactolipid transfer between the envelopes. Different diglycosyllipid synthases (DGD1, DGD2, and Chloroflexus glucosyltransferase) were introduced into the dgd1-1 mutant of Arabidopsis in fusion with N-terminal extensions (NDGD1 and NDGD2) targeting to the outer envelope. Reconstruction of DGDG synthesis in the outer envelope membrane was observed only with diglycosyllipid synthase fusion proteins carrying NDGD1, indicating that NDGD1 enables galactolipid translocation between envelopes. NDGD1 binds to phosphatidic acid (PA) in membranes and mediates PA-dependent membrane fusion in vitro. These findings provide a mechanism for the sorting and selective channeling of lipid precursors between the galactolipid pools of the two envelope membranes

Combining morphological and genomic evidence to resolve species diversity and study speciation processes of the Pallenopsis patagonica (Pycnogonida) species complex

Background: Pallenopsis patagonica (Hoek, 1881) is a morphologically and genetically variable sea spider species whose taxonomic classification is challenging. Currently, it is considered as a species complex including several genetic lineages, many of which have not been formally described as species. Members of this species complex occur on the Patagonian and Antarctic continental shelves as well as around sub-Antarctic islands. These habitats have been strongly influenced by historical large-scale glaciations and previous studies suggested that communities were limited to very few refugia during glacial maxima. Therefore, allopatric speciation in these independent refugia is regarded as a common mechanism leading to high biodiversity of marine benthic taxa in the high-latitude Southern Hemisphere. However, other mechanisms such as ecological speciation have rarely been considered or tested. Therefore, we conducted an integrative morphological and genetic study on the P. patagonica species complex to i) resolve species diversity using a target hybrid enrichment approach to obtain multiple genomic markers, ii) find morphological characters and analyze morphometric measurements to distinguish species, and iii) investigate the speciation processes that led to multiple lineages within the species complex. Results: Phylogenomic results support most of the previously reported lineages within the P. patagonica species complex and morphological data show that several lineages are distinct species with diagnostic characters. Two lineages are proposed as new species, P. aulaeturcarum sp. nov. Dömel & Melzer, 2019 and P. obstaculumsuperavit sp. nov. Dömel, 2019, respectively. However, not all lineages could be distinguished morphologically and thus likely represent cryptic species that can only be identified with genetic tools. Further, morphometric data of 135 measurements showed a high amount of variability within and between species without clear support of adaptive divergence in sympatry. Conclusions: We generated an unprecedented molecular data set for members of the P. patagonica sea spider species complex with a target hybrid enrichment approach, which we combined with extensive morphological and morphometric analyses to investigate the taxonomy, phylogeny and biogeography of this group. The extensive data set enabled us to delineate species boundaries, on the basis of which we formally described two new species. No consistent evidence for positive selection was found, rendering speciation in allopatric glacial refugia as the most likely model of speciation

Primary B Cell Lymphoma of the CNS Mimicking Anti-LGI1 Limbic Encephalitis

Limbic encephalitis is a potentially paraneoplastic type of encephalitis mainly involving the limbic system. Recently, diagnostic criteria comprising clinical presentation as well as imaging, laboratory and electrophysiological findings have been established. Here, we show that incipient primary central nervous system lymphoma can closely resemble limbic encephalitis including positive testing for anti-LGI1 antibodies illustrating the need for thorough interpretation of initial laboratory and radiologic findings and tight follow-up examinations

Interventricular septum hematoma during cineventriculography

<p>Abstract</p> <p>Background</p> <p>Intraseptal hematoma and subsequent myocardial infarction due to accidental contrast agent deposition complicating diagnostic cineventriculography is a previously undescribed complication of angiography.</p> <p>Case presentation</p> <p>A 61 year old man was admitted at intensive care unit because of unstable angina pectoris 1 hour after coronary angiography. Transthoracic contrast echocardiography showed a non-perfused area in the middle of interventricular septum with an increase of thickening up to 26 mm. Review of cineventriculography revealed contrast enhancement in the interventricular septum after contrast medium injection and a dislocation of the pigtail catheter tip. Follow up by echocardiography and MRI showed, that intramural hematoma has resolved after 6 weeks. After 8 weeks successful stent implantation in LAD was performed and after 6 month the patient had a normal LV-function without ischemic signs or septal thickening demonstrated by stressechocardiography.</p> <p>Conclusion</p> <p>A safe and mobile position of the pigtail catheter during ventriculography in the middle of the LV cavity should be ensured to avoid this potentially life-threatening complication. For assessment and absolute measurement of intramural hematoma contrast-enhanced echocardiography is more feasible than MRI and makes interchangeable results.</p