Anode Catalysts in Anion-Exchange-Membrane Electrolysis without Supporting Electrolyte: Conductivity, Dynamics, and Ionomer Degradation.

Anion-exchange-membrane water electrolyzers (AEMWEs) in principle operate without soluble electrolyte using earth-abundant catalysts and cell materials and thus lower the cost of green H2 . Current systems lack competitive performance and the durability needed for commercialization. One critical issue is a poor understanding of catalyst-specific degradation processes in the electrolyzer. While non-platinum-group-metal (non-PGM) oxygen-evolution catalysts show excellent performance and durability in strongly alkaline electrolyte, this has not transferred directly to pure-water AEMWEs. Here, AEMWEs with five non-PGM anode catalysts are built and the catalysts' structural stability and interactions with the alkaline ionomer are characterized during electrolyzer operation and post-mortem. The results show catalyst electrical conductivity is one key to obtaining high-performing systems and that many non-PGM catalysts restructure during operation. Dynamic Fe sites correlate with enhanced degradation rates, as does the addition of soluble Fe impurities. In contrast, electronically conductive Co3 O4 nanoparticles (without Fe in the crystal structure) yield AEMWEs from simple, standard preparation methods, with performance and stability comparable to IrO2 . These results reveal the fundamental dynamic catalytic processes resulting in AEMWE device failure under relevant conditions, demonstrate a viable non-PGM catalyst for AEMWE operation, and illustrate underlying design rules for engineering anode catalyst/ionomer layers with higher performance and durability.

AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines.

Though AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. Here we isolate an engineered variant, "AsCas12a Ultra", that increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We show that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications. We achieved simultaneous targeting of three clinically relevant genes in T cells at >90% efficiency and demonstrated transgene knock-in efficiencies of up to 60%. We demonstrate site-specific knock-in of a CAR in NK cells, which afforded enhanced anti-tumor NK cell recognition, potentially enabling the next generation of allogeneic cell-based therapies in oncology. AsCas12a Ultra is an advanced CRISPR nuclease with significant advantages in basic research and in the production of gene edited cell medicines.

Efficiency, Specificity and Temperature Sensitivity of Cas9 and Cas12a RNPs for DNA-free Genome Editing in Plants.

Delivery of genome editing reagents using CRISPR-Cas ribonucleoproteins (RNPs) transfection offers several advantages over plasmid DNA-based delivery methods, including reduced off-target editing effects, mitigation of random integration of non-native DNA fragments, independence of vector constructions, and less regulatory restrictions. Compared to the use in animal systems, RNP-mediated genome editing is still at the early development stage in plants. In this study, we established an efficient and simplified protoplast-based genome editing platform for CRISPR-Cas RNP delivery, and then evaluated the efficiency, specificity, and temperature sensitivity of six Cas9 and Cas12a proteins. Our results demonstrated that Cas9 and Cas12a RNP delivery resulted in genome editing frequencies (8.7-41.2%) at various temperature conditions, 22°C, 26°C, and 37°C, with no significant temperature sensitivity. LbCas12a often exhibited the highest activities, while AsCas12a demonstrated higher sequence specificity. The high activities of CRISPR-Cas RNPs at 22° and 26°C, the temperature preferred by plant transformation and tissue culture, led to high mutagenesis efficiencies (34.0-85.2%) in the protoplast-regenerated calli and plants with the heritable mutants recovered in the next generation. This RNP delivery approach was further extended to pennycress (Thlaspi arvense), soybean (Glycine max) and Setaria viridis with up to 70.2% mutagenesis frequency. Together, this study sheds light on the choice of RNP reagents to achieve efficient transgene-free genome editing in plants.

Potentiating TMEM16A does not stimulate airway mucus secretion or bronchial and pulmonary arterial smooth muscle contraction.

The calcium-activated chloride channel (CaCC) TMEM16A enables chloride secretion across several transporting epithelia, including in the airways. Additional roles for TMEM16A have been proposed, which include regulating mucus production and secretion and stimulating smooth muscle contraction. The aim of the present study was to test whether the pharmacological regulation of TMEM16A channel function, could affect any of these proposed biological roles in the airways. In vitro, neither a potent and selective TMEM16A potentiator (ETX001) nor the potent TMEM16A inhibitor (Ani9) influenced either baseline mucin release or goblet cell numbers in well-differentiated primary human bronchial epithelial (HBE) cells. In vivo, a TMEM16A potentiator was without effect on goblet cell emptying in an IL-13 stimulated goblet cell metaplasia model. Using freshly isolated human bronchi and pulmonary arteries, neither ETX001 or Ani9 had any effect on the contractile or relaxant responses of the tissues. In vivo, ETX001 also failed to influence either lung or cardiovascular function when delivered directly into the airways of telemetered rats. Together, these studies do not support a role for TMEM16A in the regulation of goblet cell numbers or baseline mucin release, or on the regulation of airway or pulmonary artery smooth muscle contraction.

Term Neonate Presenting with the Combined Occurrence of Mucolipidosis Type II and Leigh Syndrome.

Mucolipidosis II α/beta (MLII) is an autosomal recessive disease in which a gene mutation leads to improper targeting of lysosomal enzymes with an end result of accumulation of lysosomes in the mitochondria resulting in a dysfunctional mitochondria. 1 Leigh syndrome (LS) is a rare progressive neurodegenerative disorder associated with dysfunctional mitochondria and oxidative phosphorylation. 4 Both disease processes typically present in infancy. 3 7 Herein, we present a case of an infant diagnosed with both mucolipidosis II and Leigh syndrome. Genetic analysis in this case revealed two mutations (NDUFA12 c.178C > T p.Arg60* and GNPTAB c.732_733delAA) on the long arm of chromosome 12 as the etiology of MLII and LS in this neonate, respectively. We are unaware of any previously published cases of the presence of these two diseases occurring in the same patient. The complex clinical presentation of this case led to a delay in the diagnosis, and we believe that the clinical phenotypes of these two conditions were likely worsened. The genetic alterations presented in this case occurred as a result of mutations on chromosome 12. We suggest further investigation into the potential overlap in the pathophysiology, specifically the inheritance pattern, linkage disequilibrium, mitochondrial-lysosomal interaction, or crosstalk contributing to both diseases.

Lower respiratory tract delivery, airway clearance, and preclinical efficacy of inhaled GM-CSF in a postinfluenza pneumococcal pneumonia model.

Inhaled granulocyte/macrophage colony-stimulating factor (GM-CSF) shows promise as a therapeutic to treat viral and bacterial pneumonia, but no mouse model of inhaled GM-CSF has been described. We sought to 1) develop a mouse model of aerosolized recombinant mouse GM-CSF administration and 2) investigate the protection conferred by inhaled GM-CSF during influenza A virus (IAV) infection against secondary bacterial infection with pneumococcus. To assess lower respiratory tract delivery of aerosolized therapeutics, mice were exposed to aerosolized fluorescein (FITC)-labeled dextran noninvasively via an aerosolization tower or invasively using a rodent ventilator. The efficiency of delivery to the lower respiratory tracts of mice was 0.01% noninvasively compared with 0.3% invasively. The airway pharmacokinetics of inhaled GM-CSF fit a two-compartment model with a terminal phase half-life of 1.3 h. To test if lower respiratory tract levels were sufficient for biological effect, mice were infected intranasally with IAV, treated with aerosolized recombinant mouse GM-CSF, and then secondarily infected with Streptococcus pneumoniae. Inhaled GM-CSF conferred a significant survival benefit to mice against secondary challenge with S. pneumoniae (P < 0.05). Inhaled GM-CSF did not reduce airway or lung parenchymal bacterial growth but significantly reduced the incidence of S. pneumoniae bacteremia (P < 0.01). However, GM-CSF overexpression during influenza virus infection did not affect lung epithelial permeability to FITC-dextran ingress into the bloodstream. Therefore, the mechanism of protection conferred by inhaled GM-CSF appears to be locally mediated improved lung antibacterial resistance to systemic bacteremia during IAV infection.

Progress in using threonine aldolases for preparative synthesis.

Three threonine aldolases (TAs) were cloned and overexpressed in Escherichia coli (Aeromonas jandaeil-allo-threonine aldolase, E. colil-threonine aldolase and Thermotoga maritimal-allo-threonine aldolase). A Design of Experiments strategy was used to identify optimal reaction conditions for each enzyme. These conditions were used to characterize the substrate- and stereoselectivity of each TA toward a panel of aldehyde acceptors. In general, the A. jandaei TA performed best, and six representative conversions were scaled up 10-fold in order to develop downstream steps for product isolation. One key improvement was to treat the crude reaction product with Bacillus subtilis glycine oxidase, which eliminated residual starting material and significantly simplified product isolation. NMR studies were used to identify the major and minor diastereomers from the preparative-scale reactions and the absolute configurations for three representative cases.

Effect of age and vaccination on extent and spread of Chlamydia pneumoniae infection in C57BL/6 mice.

BACKGROUND: Chlamydia pneumoniae is an obligate intracellular respiratory pathogen for humans. Infection by C. pneumoniae may be linked etiologically to extra-respiratory diseases of aging, especially atherosclerosis. We have previously shown that age promotes C. pneumoniae respiratory infection and extra-respiratory spread in BALB/c mice. FINDINGS: Aged C57BL/6 mice had a greater propensity to develop chronic and/or progressive respiratory infections following experimental intranasal infection by Chlamydia pneumoniae when compared to young counterparts. A heptavalent CTL epitope minigene (CpnCTL7) vaccine conferred equal protection in the lungs of both aged and young mice. This vaccine was partially effective in protecting against C. pneumoniae spread to the cardiovascular system of young mice, but failed to provide cardiovascular protection in aged animals. CONCLUSIONS: Our findings suggest that vaccine strategies that target the generation of a C. pneumoniae-specific CTL response can protect the respiratory system of both young and aged animals, but may not be adequate to prevent dissemination of C. pneumoniae to the cardiovascular system or control replication in those tissues in aged animals.