Efficient production of the Nylon 12 monomer ω-aminododecanoic acid methyl ester from renewable dodecanoic acid methyl ester with engineered Escherichia coli

The expansion of microbial substrate and product scopes will be an important brick promoting future bioeconomy. In this study, an orthogonal pathway running in parallel to native metabolism and converting renewable dodecanoic acid methyl ester (DAME) via terminal alcohol and aldehyde to 12-aminododecanoic acid methyl ester (ADAME), a building block for the high-performance polymer Nylon 12, was engineered in Escherichia coli and optimized regarding substrate uptake, substrate requirements, host strain choice, flux, and product yield. Efficient DAME uptake was achieved by means of the hydrophobic outer membrane porin AlkL increasing maximum oxygenation and transamination activities 8.3 and 7.6-fold, respectively. An optimized coupling to the pyruvate node via a heterologous alanine dehydrogenase enabled efficient intracellular L-alanine supply, a prerequisite for self-sufficient whole-cell transaminase catalysis. Finally, the introduction of a respiratory chain-linked alcohol dehydrogenase enabled an increase in pathway flux, the minimization of undesired overoxidation to the respective carboxylic acid, and thus the efficient formation of ADAME as main product. The completely synthetic orthogonal pathway presented in this study sets the stage for Nylon 12 production from renewables. Its effective operation achieved via fine tuning the connectivity to native cell functionalities emphasizes the potential of this concept to expand microbial substrate and product scopes

MED12 regulates a transcriptional network of calcium-handling genes in the heart

The Mediator complex regulates gene transcription by linking basal transcriptional machinery with DNA-bound transcription factors. The activity of the Mediator complex is mainly controlled by a kinase submodule that is composed of 4 proteins, including MED12. Although ubiquitously expressed, Mediator subunits can differentially regulate gene expression in a tissue-specific manner. Here, we report that MED12 is required for normal cardiac function, such that mice with conditional cardiac-specific deletion of MED12 display progressive dilated cardiomyopathy. Loss of MED12 perturbs expression of calcium-handling genes in the heart, consequently altering calcium cycling in cardiomyocytes and disrupting cardiac electrical activity. We identified transcription factors that regulate expression of calcium-handling genes that are downregulated in the heart in the absence of MED12, and we found that MED12 localizes to transcription factor consensus sequences within calcium-handling genes. We showed that MED12 interacts with one such transcription factor, MEF2, in cardiomyocytes and that MED12 and MEF2 co-occupy promoters of calcium-handling genes. Furthermore, we demonstrated that MED12 enhances MEF2 transcriptional activity and that overexpression of both increases expression of calcium-handling genes in cardiomyocytes. Our data support a role for MED12 as a coordinator of transcription through MEF2 and other transcription factors. We conclude that MED12 is a regulator of a network of calcium-handling genes, consequently mediating contractility in the mammalian heart

Glial-Specific Deletion of Med12 Results in Rapid Hearing Loss via Degradation of the Stria Vascularis

Mediator protein complex subunit 12 (Med12) is a core component of the basal transcriptional apparatus and plays a critical role in the development of many tissues. Mutations in Med12 are associated with X-linked intellectual disability syndromes and hearing loss; however, its role in nervous system function remains undefined. Here, we show that temporal conditional deletion of Med12 in astrocytes in the adult central nervous system results in region specific alterations in astrocyte morphology. Surprisingly, behavioral studies revealed rapid hearing loss after adult deletion of Med12 that was confirmed by a complete abrogation of auditory brainstem responses. Cellular analysis of the cochlea revealed degeneration of the stria vascularis, in conjunction with disorganization of basal cells adjacent to the spiral ligament and downregulation of key cell adhesion proteins. Physiological analysis revealed early changes in endocochlear potential, consistent with strial-specific defects. Together, our studies reveal that Med12 regulates auditory function in the adult by preserving the structural integrity of the stria vascularis

Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells

In response to cell swelling, volume-regulated anion channels (VRACs) participate in a process known as regulatory volume decrease (RVD). Only recently, first insight into the molecular identity of mammalian VRACs was obtained by the discovery of the leucine-rich repeats containing 8A (LRRC8A) gene. Here, we show that bestrophin 1 (BEST1) but not LRRC8A is crucial for volume regulation in human retinal pigment epithelium (RPE) cells. Whole-cell patch-clamp recordings in RPE derived from human-induced pluripotent stem cells (hiPSC) exhibit an outwardly rectifying chloride current with characteristic functional properties of VRACs. This current is severely reduced in hiPSC-RPE cells derived from macular dystrophy patients with pathologic BEST1 mutations. Disruption of the orthologous mouse gene (Best1−/−) does not result in obvious retinal pathology but leads to a severe subfertility phenotype in agreement with minor endogenous expression of Best1 in murine RPE but highly abundant expression in mouse testis. Sperm from Best1−/− mice showed reduced motility and abnormal sperm morphology, indicating an inability in RVD. Together, our data suggest that the molecular identity of VRACs is more complex—that is, instead of a single ubiquitous channel, VRACs could be formed by cell type- or tissue-specific subunit composition. Our findings provide the basis to further examine VRAC diversity in normal and diseased cell physiology, which is key to exploring novel therapeutic approaches in VRAC-associated pathologies

Post-stroke inhibition of induced NADPH oxidase type 4 prevents oxidative stress and neurodegeneration

Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress. However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (Nox4(-/-)) of either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age, as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in Nox4(-/-) mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source of oxidative stress and novel class of drug target for stroke therapy

Investigation of sex-specific effects of apolipoprotein E on severity of EAE and MS

Background Despite pleiotropic immunomodulatory effects of apolipoprotein E (apoE) in vitro, its effects on the clinical course of experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS) are still controversial. As sex hormones modify immunomodulatory apoE functions, they may explain contentious findings. This study aimed to investigate sex-specific effects of apoE on disease course of EAE and MS. Methods MOG35-55 induced EAE in female and male apoE-deficient mice was assessed clinically and histopathologically. apoE expression was investigated by qPCR. The association of the MS severity score (MSSS) and APOE rs429358 and rs7412 was assessed across 3237 MS patients using linear regression analyses. Results EAE disease course was slightly attenuated in male apoE-deficient (apoE −/− ) mice compared to wildtype mice (cumulative median score: apoE −/−  = 2 [IQR 0.0–4.5]; wildtype = 4 [IQR 1.0–5.0]; n = 10 each group, p = 0.0002). In contrast, EAE was more severe in female apoE −/− mice compared to wildtype mice (cumulative median score: apoE −/−  = 3 [IQR 2.0–4.5]; wildtype = 3 [IQR 0.0–4.0]; n = 10, p = 0.003). In wildtype animals, apoE expression during the chronic EAE phase was increased in both females and males (in comparison to naïve animals; p < 0.001). However, in MS, we did not observe a significant association between MSSS and rs429358 or rs7412, neither in the overall analyses nor upon stratification for sex. Conclusions apoE exerts moderate sex-specific effects on EAE severity. However, the results in the apoE knock-out model are not comparable to effects of polymorphic variants in the human APOE gene, thus pinpointing the challenge of translating findings from the EAE model to the human disease

A mouse model for Sorsby fundus dystrophy

purpose. Sorsby fundus dystrophy (SFD) is a rare, late-onset macular dystrophy caused by mutations in the tissue inhibitor of metalloproteinases-3 (TIMP3) gene. The known mutations introduce potentially unpaired cysteine residues in the C terminus of the protein and result in the formation of higher-molecular-weight protein complexes of as yet unknown composition and functional consequences in the pathologic course of SFD. To facilitate in vivo investigation of mutant TIMP3, the authors generated a knock-in mouse carrying a disease-related Ser156Cys mutation in the orthologous murine Timp3 gene. methods. Site-directed mutagenesis and homologous recombination in embryonic stem (ES) cells was used to generate mutant ES cells carrying the Timp3 S156C allele. Chimeric animals were obtained, of which two displayed germline transmission of the mutated allele. Molecular genetic, biochemical, electron microscopic, and electrodiagnostic techniques were used for characterization. results. At 8 months of age, knock-in mice showed abnormalities in the inner aspect of Bruch’s membrane and in the organization of the adjacent basal microvilli of the retinal pigment epithelium (RPE). Changes resembling those in the mutant animals were also present to some extent in normal littermates, but only at an advanced age of 30 months. Long-term electrodiagnostic recordings indicated normal retinal function throughout life. The biochemical characteristics of the mutant protein appear similar in humans and knock-in mice, suggesting common molecular pathways in the two species. The localization of the mutant protein in the eye is normal, although there is evidence of increased Timp3 levels in Bruch’s membrane of mutant animals. conclusions. The knock-in mice display early features of age-related changes in Bruch’s membrane and the RPE that may represent the primary clinical manifestations of SFD. In addition, our immunolabeling studies and biochemical data support a model proposing that site-specific excess rather than absence or deficiency of functional Timp3 may be the primary consequence of the known Timp3 mutations

Low catalytic activity is insufficient to induce disease pathology in triosephosphate isomerase (TPI) deficiency

Triosephosphate isomerase (TPI) deficiency is a fatal genetic disorder characterized by hemolytic anemia and neurological dysfunction. Although the enzyme defect in TPI was discovered in the 1960s, the exact etiology of the disease is still debated. Some aspects indicate the disease could be caused by insufficient enzyme activity, whereas other observations indicate it could be a protein misfolding disease with tissue-specific differences in TPI activity. We generated a mouse model in which exchange of a conserved catalytic amino acid residue (isoleucine to valine, Ile170Val) reduces TPI specific activity without affecting the stability of the protein dimer. TPIIle170Val/Ile170Val mice exhibit an approximately 85% reduction in TPI activity consistently across all examined tissues, which is a stronger average, but more consistent, activity decline than observed in patients or symptomatic mouse models that carry structural defect mutant alleles. While monitoring protein expression levels revealed no evidence for protein instability, metabolite quantification indicated that glycolysis is affected by the active site mutation. TPIIle170Val/Ile170Val mice develop normally and show none of the disease symptoms associated with TPI deficiency. Therefore, without the stability defect that affects TPI activity in a tissue-specific manner, a strong decline in TPI catalytic activity is not sufficient to explain the pathological onset of TPI deficiency

Closed-loop control of product properties in metal forming

Metal forming processes operate in conditions of uncertainty due to parameter variation and imperfect understanding. This uncertainty leads to a degradation of product properties from customer specifications, which can be reduced by the use of closed-loop control. A framework of analysis is presented for understanding closed-loop control in metal forming, allowing an assessment of current and future developments in actuators, sensors and models. This leads to a survey of current and emerging applications across a broad spectrum of metal forming processes, and a discussion of likely developments.Engineering and Physical Sciences Research Council (Grant ID: EP/K018108/1)This is the final version of the article. It first appeared from Elsevier via https://doi.org/10.1016/j.cirp.2016.06.00