Engineering NADH metabolism in Saccharomyces cerevisiae: formate as an electron donor for glycerol production by anaerobic, glucose-limited chemostat cultures

Anaerobic Saccharomyces cerevisiae cultures reoxidize the excess NADH formed in biosynthesis via glycerol production. This study investigates whether cometabolism of formate, a well-known NADH-generating substrate in aerobic cultures, can increase glycerol production in anaerobic S. cerevisiae cultures. In anaerobic, glucose-limited chemostat sultures (D=0.10 h(-1)) with molar formate-to-glucose ratios of 0 to 0.5, only a small fraction of the formate added to the cultures was consumed. To investigate whether incomplete formate consumption was by the unfavourable kinetics of yeast formate dehydrogenase (high k(M) for formate at low intracellular NAD(+) concentrations) strains were constructed in which the FDH1 and/or GPD2 genes, encoding formate dehydrogenase and glycerol-3-phosphate dehydrogenase, respectively, were overexpressed. The engineered strains consumed up to 70% of the formate added to the feed, thereby increasing glycerol yields to 0.3 mol mol(-1) glucose at a formate-to-glucose ratio of 0.34. In all strains tested, the molar ratio between formate consumption and additional glycerol production relative to a reference culture equalled one. While demonstrating that that format can be use to enhance glycerol yields in anaerobic S. cerevisiae cultures, This study also reveals kinetic constraints of yeast formate dehydrogenase as an NADH-generating system in yeast mediated reduction processes.     

New insights in the cellular processing of platinum antitumor compounds, using fluorophore-labeled platinum complexes and digital fluorescence microscopy

The cellular distribution and processing pathways of two platinum compounds, modeling the antitumor drug cisplatin (cDDP) in human osteosarcoma (U2-OS) cells is reported. A [Pt(en)Cl] entity has been covalently linked to a carboxyfluorescein diacetate (CFDA) moiety and to a dinitrophenyl (DNP) moiety. The two different constructs were administered to living cell cultures that were analyzed using digital fluorescence microscopy. The non-fluorescent CFDA construct becomes fluorescent after cellular uptake and subsequent acetate hydrolysis by esterases, and is therefore suitable to monitor platinum in living cells; the DNP construct can be visualized by immunocytochemistry and consequently serves as a control. Both complexes were readily internalized by the cells, and localized throughout the whole cell. After 2-3 h the complex accumulated in the nucleus, but 6-8 h after incubation a punctuate staining of a cytoplasmic region was observed, that persisted and became more pronounced after 24 h. The overall fluorescence in the cell decreased over time, implying a secretion of the platinum complex. Surprisingly, the accumulation remained visible after 72 h. Co-localization experiments with a Golgi apparatus-selective stain indicate the involvement of Golgi vesicles in intracellular processing of cisplatin-derived complexes. Immunocytochemical studies, using the DNP derivative, resulted in very similar images as obtained with the CFDA construct. CFDA-boc (a non-platinum-containing fluorescein derivative) was used as control: a faint staining throughout the whole cell was observed. Cisplatin-resistant U2-OS/Pt cells showed staining patterns very similar to the U2-OS cells using both platinum constructs. This study illustrates that only a very small portion of the platinum complex eventually remains bound to DNA, as after 24 h no significant fluorescence could be observed in the nucleus. Cisplatin-derived complexes with fluorescent tags afford a new insight into the cellular processing of these complexes and therefore may contribute to further unraveling of the mechanism of platinum antitumor complexes.     

Invasive surgery reduces infarct size and preserves cardiac function in a porcine model of myocardial infarction

Reperfusion injury following myocardial infarction (MI) increases infarct size (IS) and deteriorates cardiac function. Cardioprotective strategies in large animal MI models often failed in clinical trials, suggesting translational failure. Experimentally, MI is induced artificially and the effect of the experimental procedures may influence outcome and thus clinical applicability. The aim of this study was to investigate if invasive surgery, as in the common open chest MI model affects IS and cardiac function. Twenty female landrace pigs were subjected to MI by transluminal balloon occlusion. In 10 of 20 pigs, balloon occlusion was preceded by invasive surgery (medial sternotomy). After 72 hrs, pigs were subjected to echocardiography and Evans blue/triphenyl tetrazoliumchloride double staining to determine IS and area at risk. Quantification of IS showed a significant IS reduction in the open chest group compared to the closed chest group (IS versus area at risk: 50.9 ± 5.4% versus 69.9 ± 3.4%, P = 0.007). End systolic LV volume and LV ejection fraction measured by echocardiography at follow‐up differed significantly between both groups (51 ± 5 ml versus 65 ± 3 ml, P = 0.033; 47.5 ± 2.6% versus 38.8 ± 1.2%, P = 0.005). The inflammatory response in the damaged myocardium did not differ between groups. This study indicates that invasive surgery reduces IS and preserves cardiac function in a porcine MI model. Future studies need to elucidate the effect of infarct induction technique on the efficacy of pharmacological therapies in large animal cardioprotection studies.     

Biallelic Mutations in UNC80 Cause Persistent Hypotonia,Encephalopathy, Growth Retardation,and Severe Intellectual Disability

Ion channel proteins are required for both the establishment of resting membrane potentials and the generation of action potentials. Hundreds of mutations in genes encoding voltage-gated ion channels responsible for action potential generation have been found to cause severe neurological diseases. In contrast, the roles of voltage-independent “leak” channels, important for the establishment and maintenance of resting membrane potentials upon which action potentials are generated, are not well established in human disease. UNC80 is a large component of the NALCN sodium-leak channel complex that regulates the basal excitability of the nervous system. Loss-of-function mutations of NALCN cause infantile hypotonia with psychomotor retardation and characteristic facies (IHPRF). We report four individuals from three unrelated families who have homozygous missense or compound heterozygous truncating mutations in UNC80 and persistent hypotonia, encephalopathy, growth failure, and severe intellectual disability. Compared to control cells, HEK293T cells transfected with an expression plasmid containing the c.5098C>T (p.Pro1700Ser) UNC80 mutation found in one individual showed markedly decreased NALCN channel currents. Our findings demonstrate the fundamental significance of UNC80 and basal ionic conductance to human health.     

Mortality Patterns in Patients with Multiple Trauma: A Systematic Review of Autopsy Studies

Purpose

A high percentage (50%-60%) of trauma patients die due to their injuries prior to arrival at the hospital. Studies on preclinical mortality including post-mortem examinations are rare. In this review, we summarized the literature focusing on clinical and preclinical mortality and studies included post-mortem examinations.

Methods

A literature search was conducted using PubMed/Medline database for relevant medical literature in English or German language published within the last four decades (1980–2015). The following MeSH search terms were used in different combinations: “multiple trauma”, “epidemiology”, “mortality “, “cause of death”, and “autopsy”. References from available studies were searched as well.

Results

Marked differences in demographic parameters and injury severity between studies were identified. Moreover, the incidence of penetrating injuries has shown a wide range (between 4% and 38%). Both unimodal and bimodal concepts of trauma mortality have been favored. Studies have shown a wide variation in time intervals used to analyze the distribution of death. Thus, it is difficult to say which distribution is correct.

Conclusions

We have identified variable results indicating bimodal or unimodal death distribution. Further more stundardized studies in this field are needed. We would like to encourage investigators to choose the inclusion criteria more critically and to consider factors affecting the pattern of mortality.     

Reaction of DNA oligonucleotides with [Pt(dien)GSMe]2+ (GSMe =S-methylated glutathione) and cis-[Pt(NH3)2(GSMe)2]2+: evidence of oligonucleotide platination via sulfur-coordinated platinum intermediates

To study the possibility of DNA platination via platinum-sulfur coordinated intermediates, the reactions of the complexes [Pt(dien)GSMe]2+ (GSMe=S-methylated glutathione) and cis-[Pt(NH3)2(GSMe)2]2+ with the synthetic oligonucleotides d(ATATGCATAT), d(ATTACCGGTAAT), and d(ATCCTATTTTTTTTAGGAT) have been investigated. The reactions were studied using FPLC, NMR, and mass spectrometry. It was found that the sulfur atom of the platinum-thioether adduct is substituted by these oligonucleotides. For the reactions with [Pt(dien)GSMe]2+ at 310 K, half-lives were determined to be t 1/2 =147+/-7 h for d(ATATGCATAT), t 1/2 =84+/-4 h) for d(ATTACCGGTAAT), and t 1/2 = 21+/-1 h for d(ATCCTATTTTTTTTAGGAT. This study clearly shows that it is indeed possible for oligonucleotides to be platinated via Pt-thioether coordinated intermediates. The rates at which such substitutions occur, however, makes it improbable that such a mechanism contributes significantly to the antitumor activity of cisplatin.     

Physiological and genetic engineering of cytosolic redox metabolism in Saccharomyces cerevisiae for improved glycerol production

Previous metabolic engineering strategies for improving glycerol production by Saccharomyces cerevisiae were constrained to a maximum theoretical glycerol yield of 1 mol.(molglucose)(-1) due to the introduction of rigid carbon, ATP or redox stoichiometries. In the present study, we sought to circumvent these constraints by (i) maintaining flexibility at fructose-1,6-bisphosphatase and triosephosphate isomerase, while (ii) eliminating reactions that compete with glycerol formation for cytosolic NADH and (iii) enabling oxidative catabolism within the mitochondrial matrix. In aerobic, glucose-grown batch cultures a S. cerevisiae strain, in which the pyruvate decarboxylases the external NADH dehydrogenases and the respiratory chain-linked glycerol-3-phosphate dehydrogenase were deleted for this purpose, produced glycerol at a yield of 0.90 mol.(molglucose)(-1). In aerobic glucose-limited chemostat cultures, the glycerol yield was ca. 25% lower, suggesting the involvement of an alternative glucose-sensitive mechanism for oxidation of cytosolic NADH. Nevertheless, in vivo generation of additional cytosolic NADH by co-feeding of formate to aerobic, glucose-limited chemostat cultures increased the glycerol yield on glucose to 1.08 mol mol(-1). To our knowledge, this is the highest glycerol yield reported for S. cerevisiae.     

Malic acid production by Saccharomyces cerevisiae: engineering of pyruvate carboxylation, oxaloacetate reduction, and malate export

Malic acid is a potential biomass-derivable "building block" for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production from glucose proceeds via carboxylation of pyruvate, followed by reduction of oxaloacetate to malate. This redox- and ATP-neutral, CO(2)-fixing pathway has a theoretical maximum yield of 2 mol malate (mol glucose)(-1). A previously engineered glucose-tolerant, C(2)-independent pyruvate decarboxylase-negative S. cerevisiae strain was used as the platform to evaluate the impact of individual and combined introduction of three genetic modifications: (i) overexpression of the native pyruvate carboxylase encoded by PYC2, (ii) high-level expression of an allele of the MDH3 gene, of which the encoded malate dehydrogenase was retargeted to the cytosol by deletion of the C-terminal peroxisomal targeting sequence, and (iii) functional expression of the Schizosaccharomyces pombe malate transporter gene SpMAE1. While single or double modifications improved malate production, the highest malate yields and titers were obtained with the simultaneous introduction of all three modifications. In glucose-grown batch cultures, the resulting engineered strain produced malate at titers of up to 59 g liter(-1) at a malate yield of 0.42 mol (mol glucose)(-1). Metabolic flux analysis showed that metabolite labeling patterns observed upon nuclear magnetic resonance analyses of cultures grown on (13)C-labeled glucose were consistent with the envisaged nonoxidative, fermentative pathway for malate production. The engineered strains still produced substantial amounts of pyruvate, indicating that the pathway efficiency can be further improved.