Co-production of hydrogen and ethyl acetate in Escherichia coli

Background Ethyl acetate (C4H8O2) and hydrogen (H2) are industrially relevant compounds that preferably are produced via sustainable, non-petrochemical production processes. Both compounds are volatile and can be produced by Escherichia coli before. However, relatively low yields for hydrogen are obtained and a mix of by-products renders the sole production of hydrogen by micro-organisms unfeasible. High yields for ethyl acetate have been achieved, but accumulation of formate remained an undesired but inevitable obstacle. Coupling ethyl acetate production to the conversion of formate into H2 may offer an interesting solution to both drawbacks. Ethyl acetate production requires equimolar amounts of ethanol and acetyl-CoA, which enables a redox neutral fermentation, without the need for production of by-products, other than hydrogen and CO2. Results We engineered Escherichia coli towards improved conversion of formate into H2 and CO2 by inactivating the formate hydrogen lyase repressor (hycA), both uptake hydrogenases (hyaAB, hybBC) and/or overexpressing the hydrogen formate lyase activator (fhlA), in an acetate kinase (ackA) and lactate dehydrogenase (ldhA)-deficient background strain. Initially 10 strains, with increasing number of modifications were evaluated in anaerobic serum bottles with respect to growth. Four reference strains ΔldhAΔackA, ΔldhAΔackA p3-fhlA, ΔldhAΔackAΔhycAΔhyaABΔhybBC and ΔldhAΔackAΔhycAΔhyaABΔhybBC p3-fhlA were further equipped with a plasmid carrying the heterologous ethanol acyltransferase (Eat1) from Wickerhamomyces anomalus and analyzed with respect to their ethyl acetate and hydrogen co-production capacity. Anaerobic co-production of hydrogen and ethyl acetate via Eat1 was achieved in 1.5-L pH-controlled bioreactors. The cultivation was performed at 30 °C in modified M9 medium with glucose as the sole carbon source. Anaerobic conditions and gas stripping were established by supplying N2 gas. Conclusions We showed that the engineered strains co-produced ethyl acetate and hydrogen to yields exceeding 70% of the pathway maximum for ethyl acetate and hydrogen, and propose in situ product removal via gas stripping as efficient technique to isolate the products of interest.

Genotypic analysis of the female BPH/5 mouse, a model of superimposed preeclampsia

Animal models that recapitulate human diseases and disorders are widely used to investigate etiology, diagnosis, and treatment of those conditions in people. Disorders during pregnancy are particularly difficult to explore as interventions in pregnant women are not easily performed. Therefore, models that allow for pre-conception investigations are advantageous for elucidating the mechanisms involved in adverse pregnancy outcomes that are responsible for both maternal and fetal morbidity, such as preeclampsia. The Blood Pressure High (BPH)/5 mouse model has been used extensively to study the pathogenesis of preeclampsia. The female BPH/5 mouse is obese with increased adiposity and borderline hypertension, both of which are exacerbated with pregnancy making it a model of superimposed preeclampsia. Thus, the BPH/5 model shares traits with a large majority of women with pre-existing conditions that predisposes them to preeclampsia. We sought to explore the genome of the BPH/5 female mouse and determine the genetic underpinnings that may contribute to preeclampsia-associated phenotypes in this model. Using a whole genome sequencing approach, we are the first to characterize the genetic mutations in BPH/5 female mice that make it unique from the closely related BPH/2 model and the normotensive background strain, C57Bl/6. We found the BPH/5 female mouse to be uniquely different from BPH/2 and C57Bl/6 mice with a genetically complex landscape. The majority of non-synonymous consequences within the coding region of BPH/5 females were missense mutations found most abundant on chromosome X when comparing BPH/5 and BPH/2, and on chromosome 8 when comparing BPH/5 to C57Bl/6. Genetic mutations in BPH/5 females largely belong to immune system-related processes, with overlap between BPH/5 and BPH/2 models. Further studies examining each gene mutation during pregnancy are warranted to determine key contributors to the BPH/5 preeclamptic-like phenotype and to identify genetic similarities to women that develop preeclampsia.

Longitudinal characterization of neuroanatomical changes in the Fischer 344 rat brain during normal aging and between sexes

Animal models are widely used to study the pathophysiology of disease and to evaluate the efficacy of novel interventions, crucial steps towards improving disease outcomes in humans. The Fischer 344 (F344) wildtype rat is a common experimental background strain for transgenic models of disease and is also one of the most frequently used models in aging research. Despite frequency of use, characterization of neuroanatomical change with age has not been performed in the F344 rat. To this end, we present a comprehensive longitudinal examination of morphometric change in 73 brain regions and at a voxel-wise level during normative aging in a mixed-sex cohort of F344 rats. We identified age- and sex-related changes in regions such as the cortex, hippocampus, cingulum, caudoputamen, and nucleus accumbens, which are implicated in memory and motor control circuits frequently affected by aging and neurodegenerative disease. These findings provide a baseline for neuroanatomical changes associated with aging in male and female F344 rats, to which data from transgenic models or other background strains can be compared.

Challenges in predicting Greenland supraglacial lake drainages at the regional scale

A leading hypothesis for the mechanism of fast supraglacial lake drainages is that transient extensional stresses briefly allow crevassing in otherwise compressional ice flow regimes. Lake water can then hydrofracture a crevasse to the base of the ice sheet, and river inputs can maintain this connection as a moulin. If future ice sheet models are to accurately represent moulins, we must understand their formation processes, timescales, and locations. Here, we use remote-sensing velocity products to constrain the relationship between strain rates and lake drainages across ∼ 1600 km2 in Pâkitsoq, western Greenland, between 2002–2019. We find significantly more extensional background strain rates at moulins associated with fast-draining lakes than at slow-draining or non-draining lake moulins. We test whether moulins in more extensional background settings drain their lakes earlier, but we find insignificant correlation. To investigate the frequency at which strain-rate transients are associated with fast lake drainage, we examined Landsat-derived strain rates over 16 and 32 d periods at moulins associated with 240 fast-lake-drainage events over 18 years. A low signal-to-noise ratio, the presence of water, and the multi-week repeat cycle obscured any resolution of the hypothesized transient strain rates. Our results support the hypothesis that transient strain rates drive fast lake drainages. However, the current generation of ice sheet velocity products, even when stacked across hundreds of fast lake drainages, cannot resolve these transients. Thus, observational progress in understanding lake drainage initiation will rely on field-based tools such as GPS networks and photogrammetry.

Numerical modelling of quartz-biotite aggregates: Insights on strain weakening and two-phase flow laws

<div> <div> <div> <p>Strain weakening is a prerequisite for localization of strain and therefore crucial for the understanding of shear zone evolution. In the context of progressive deformation of multi-phase aggregates, it is unclear whether the change in geometry and orientation of the involved phases leads to structural or geometric strain weakening and thus may control strain localization. Consequently, the question arises how the ductile flow of two-phase rocks can be described or determined. To contribute to a better understanding of the knowledge gaps outlined above, two-dimensional numerical shear experiments of quartz-biotite aggregates were conducted at varying temperatures, background strain rates and fluid pressure ratios. Textural variations after a shear strain of <em>γ </em>≈ 10 appear to be dependent on the viscosity contrast between the minerals involved. To estimate whether a numerical experiment is undergoing strain weakening or strain hardening (or both), the temporal evolution of the mean second invariant of the deviatoric stress tensor was tracked. The results suggest that strain weakening occurs if biotite-inclusions are distinctly isolated and that it is more effective under conditions with larger viscosity contrasts between matrix and inclusions. However, the stress drops in numerical experiments with purely structural / textural strain weakening are rather low (−1.1 to −6.4%) compared to other strain weakening processes. It appears that phase rearrangement and change in phase geometry with evolving strain is of minor importance for the occurrence of strain weakening. Based on the numerical experiments and assuming a power-law relationship between stress and strain, the flow-law parameters of quartz-biotite aggregates with different biotite contents were determined. The results are in the range of existing experimental and analytical mixed-aggregates flow-laws. However, the variations between the different flow-laws show that further research is required, for which numerical models as used in the present study could serve as basis.</p> </div> </div> </div>

Detection of Genotype-Specific Antibody Responses to Glycoproteins B and H in Primary and Non-Primary Human Cytomegalovirus Infections by Peptide-Based ELISA

Background: Strain-specific antibodies to human cytomegalovirus (HCMV) glycoproteins B and H (gB and gH) have been proposed as a potential diagnostic tool for identifying reinfection. We investigated genotype-specific IgG antibody responses in parallel with defining the gB and gH genotypes of the infecting viral strains. Methods: Subjects with primary (n = 20) or non-primary (n = 25) HCMV infection were studied. The seven gB (gB1-7) and two gH (gH1-2) genotypes were determined by real-time PCR and whole viral genome sequencing, and genotype-specific IgG antibodies were measured by a peptide-based enzyme-linked immunosorbent assay (ELISA). Results: Among subjects with primary infection, 73% (n = 8) infected by gB1-HCMV and 63% (n = 5) infected by gB2/3-HCMV had genotype-specific IgG antibodies to gB (gB2 and gB3 are similar in the region tested). Peptides from the rarer gB4-gB7 genotypes had nonspecific antibody responses. All subjects infected by gH1-HCMV and 86% (n = 6) infected by gH2-HCMV developed genotype-specific responses. Among women with non-primary infection, gB and gH genotype-specific IgG antibodies were detected in 40% (n = 10) and 80% (n = 20) of subjects, respectively. Conclusions: Peptide-based ELISA is capable of detecting primary genotype-specific IgG responses to HCMV gB and gH, and could be adopted for identifying reinfections. However, about half of the subjects did not have genotype-specific IgG antibodies to gB.

Pneumococcal genetic variability influences age-dependent bacterial carriage

The pneumococcal conjugate vaccine (PCV) primarily reduces disease burden in adults through a reduction in carriage prevalence of invasive serotypes in children. Current vaccine formulations are the same for both adults and children, but tailoring these formulations to age category could optimize vaccine efficacy. Identification of specific pneumococcal genetic factors associated with carriage in younger or older age groups may suggest alternative formulations and contribute to a better mechanistic understanding of immunity. Here, we used whole genome sequencing to dissect pneumococcal variation associated with age. We performed genome sequencing in a large carriage cohort, and conducted a meta-analysis with an existing carriage study. We compiled a dictionary of pathogen genetic variation including serotype, sequence cluster, sequence elements, SNPs, burden combined rare variants, and clusters of orthologous genes (COGs) for each cohort – all of which used in a genome-wide association with host age. Age-dependent colonization had some heritability, though this varied between cohorts (h2 = 0.10, 0.00 – 0.69 95% CI in the first; h2 = 0.46, 0.33 – 0.60 95% CI in the second cohort). We found that serotypes and genetic background (strain) explained most of the heritability in each cohort (h2serotype = 0.06 and h2GPSC = 0.04 in the first; h2serotype = 0.20 and h2GPSC = 0.23 in the second cohort). We found one candidate association (p = 1.2×10−9) upstream of an accessory Sec-dependent serine-rich glycoprotein adhesin. Overall, association with age was highly cohort and strain dependent, supporting proposals for a future vaccination strategy which is primarily targeted using serotypes rather than proteins, and is tailored towards specific pathogen populations.

Minimal gene set from Sinorhizobium (Ensifer) meliloti pSymA required for efficient symbiosis with Medicago

Reduction of N2 gas to ammonia in legume root nodules is a key component of sustainable agricultural systems. Root nodules are the result of a symbiosis between leguminous plants and bacteria called rhizobia. Both symbiotic partners play active roles in establishing successful symbiosis and nitrogen fixation: while root nodule development is mostly controlled by the plant, the rhizobia induce nodule formation, invade, and perform N2 fixation once inside the plant cells. Many bacterial genes involved in the rhizobia–legume symbiosis are known, and there is much interest in engineering the symbiosis to include major nonlegume crops such as corn, wheat, and rice. We sought to identify and combine a minimal bacterial gene complement necessary and sufficient for symbiosis. We analyzed a model rhizobium, Sinorhizobium (Ensifer) meliloti, using a background strain in which the 1.35-Mb symbiotic megaplasmid pSymA was removed. Three regions representing 162 kb of pSymA were sufficient to recover a complete N2-fixing symbiosis with alfalfa, and a targeted assembly of this gene complement achieved high levels of symbiotic N2 fixation. The resulting gene set contained just 58 of 1,290 pSymA protein-coding genes. To generate a platform for future synthetic manipulation, the minimal symbiotic genes were reorganized into three discrete nod, nif, and fix modules. These constructs will facilitate directed studies toward expanding the symbiosis to other plant partners. They also enable forward-type approaches to identifying genetic components that may not be essential for symbiosis, but which modulate the rhizobium’s competitiveness for nodulation and the effectiveness of particular rhizobia–plant symbioses.

Strain- and age-dependent features of the nigro-striatal circuit in three common laboratory mouse strains, C57BL/6J, A/J, and DBA/2J - Implications for Parkinson’s disease modeling

Mouse models have been instrumental in understanding genetic determinants of aging and its crucial role in neurodegenerative diseases. However, few studies have analyzed the evolution of the mouse brain over time at baseline. Furthermore, mouse brain studies are commonly conducted on the C57BL/6 strain, limiting the analysis to a specific genetic background. In Parkinson’s disease, the gradual demise of nigral dopaminergic neurons mainly contributes to the motor symptoms. Interestingly, a decline of the dopaminergic neuron function and integrity is also a characteristic of physiological aging in some species. Age-related nigro-striatal features have never been studied in mice of different genetic backgrounds. In this study, we analyze the morphological features in the striatum of three common mouse strains, C57BL/6J, A/J, and DBA/2J at 3-, 9- and 15 months of age. By measuring dopaminergic markers, we uncover age-related changes that differ between strains and evolve dynamically over time. Overall, our results highlight the importance of considering background strain and age when studying the murine nigro-striatal circuit in health and disease.HighlightsStudy of the integrity of the nigro-striatal circuit in C57BL/6J, A/J, and DBA/2J at different agesAge related evolution of essential features of nigral dopaminergic neurons differ between strainsConsider background strain and age is crutial to study the nigrostriatal circuit in health and disease

Challenges in predicting Greenland supraglacial lake drainages at the regional scale

A leading hypothesis for the mechanism of fast supraglacial lake drainages is that transient extensional stresses briefly allow crevassing in otherwise-compressive ice flow regimes. Lake water can then hydrofracture the crevasse to the base of the ice sheet, and river inputs can maintain this connection as a moulin. If future ice-sheet models are to accurately represent moulins, we must understand their formation processes, timescales, and locations. Here, we use remote-sensing velocity products to constrain the relationship between strain rates and lake drainages across ~ 1600 km2 in Pâkitsoq, western Greenland, between 2002–2019. We find significantly more-extensional background strain rates at moulins associated with fast-draining lakes than at slow-draining or non-draining lake moulins. We test whether moulins in more-extensional background settings drain their lakes earlier, but find insignificant correlation. To investigate the frequency that strain-rate transients are associated with fast lake drainage, we examined Landsat-derived strain rates over 16- and 32-day periods at moulins associated with 240 fast lake drainage events over 18 years. A low signal-to-noise ratio, the presence of water, and the multi-week repeat cycle obscured any resolution of the hypothesized transient strain rates. Our results support the hypothesis that transient strain rates drive fast lake drainages. However, the current generation of ice-sheet velocity products, even when stacked across hundreds of fast lake drainages, cannot resolve these transients. Thus, observational progress in understanding lake drainage initiation will rely on field-based tools such as GPS networks and photogrammetry.