Engineering the bilayer: Emerging genetic tool kits for mechanistic lipid biology

The structural diversity of lipids underpins the biophysical properties of cellular membranes, which vary across all scales of biological organization. Because lipid composition results from complex metabolic and transport pathways, its experimental control has been a major goal of mechanistic membrane biology. Here, we argue that in the wake of synthetic biology, similar metabolic engineering strategies can be applied to control the composition, physicochemical properties, and function of cell membranes. In one emerging area, titratable expression platforms allow for specific and genome-wide alterations in lipid biosynthetic genes, providing analog control over lipidome stoichiometry in membranes. Simultaneously, heterologous expression of biosynthetic genes and pathways has allowed for gain-of-function experiments with diverse lipids in non-native systems. Finally, we highlight future directions for tool development, including recently discovered lipid transport pathways to intracellular lipid pools. Further tool development providing synthetic control of membrane properties can allow biologists to untangle membrane lipid structure-associated functions.     

Rethinking protein aggregation and drug discovery in neurodegenerative diseases: Why we need to embrace complexity?

More than a century has passed since pathological protein aggregates were first identified in the brains of patients with neurodegenerative diseases (NDDs). Yet, we still do not have effective therapies to treat or slow the progression of these devastating diseases or diagnostics for early detection and monitoring disease progression. Herein, I reflect on recent findings that are challenging traditional views about the composition, ultrastructural properties, and diversity of protein pathologies in the brain, their mechanisms of formation and how we investigate and model pathological aggregation processes in the laboratory today. This article is an invitation to embrace the complexity of proteinopathies as an essential step to understanding the molecular mechanisms underpinning NDDs and to advance translational research and drug discovery in NDDs.     

Dietary practices,site-specific tumors,and age-at-death relationships

When rats are given freedom of dietary choice, the quantity and composition of the diet selected varies from animal to animal, but the risk to the individual of developing a variety of neoplasms is greatly increased over that of rats fed fixed diets. The increase in frequency of tumors cannot be explained on the basis of the amount or the composition of the diet selected. However, the application of “variable selection” statistical procedures shows that there are combinations of temporal-specific interacting dietary and dietary-dependent variables that are closely linked with cancer mortality. The multi-variable statistical model for animals dying with one type of tumor is not applicable to those dying with tumors of other types or sites. In every case, the only data required to characterize the dates of death of tumor-bearing individuals are those describing the dietary practices and growth responses prior to maturity. The model for animals with pancreatic tumors is used to illustrate the extent to which dietary practices of early life are linked with the animals’ survival.     

Fusarium asiaticum: an Emerging Pathogen Jeopardizing Postharvest Asparagus Spears

Asparagus spears are usually vulnerable to pathogenic micro‐organisms. In this study, 217 pathogens were isolated from symptomatic asparagus, and one highly virulent fungus (designated EXAP‐08) isolated from the rotted asparagus spears in cold storage was characterized in detail. Koch's postulates were checked through pathogenicity tests, indicating that EXAP‐08 infection could cause reproducible rot symptoms similar to those observed on naturally infected asparagus spears, and the pathogenicity of EXAP‐08 was also relatively higher than other Fusarium pathogens, especially at 4°C. Through morphological and molecular identification, EXAP‐08 was characterized as Fusarium asiaticum. This identification was further confirmed by phylogenetic analysis with the Histone gene H3 of EXAP‐08 and other Fusarium species. EXAP‐08 also belongs to 3A‐DON (3‐acetyl‐4‐deoxynivalenol) chemo‐type, and the mycotoxin was detected during the infection of plant, implying the potential risks of mycotoxin contamination in fresh crops infected by this pathogen. Thus, this emerging pathogen threatening edible safety of asparagus spears should deserve particular quarantine inspection in the future.     

A systems level engineered E. coli capable of efficiently producing L-phenylalanine

The biosynthesis of L-phenylalanine (Phe) is one of the most complicated amino acid synthesis pathways. In this study, the engineering of Phe producer was carried out to illustrate the effectiveness of systems level engineering: (1) inactivated glucose specific phosphoenolpyruvate-carbohydrate phosphotransferase (PTS) system by inactivation of crr to moderate the glucose uptake rate to reduce overflow metabolism; (2) genetic switch on or off the expression of phe^fbr, aroG15, ydiB, aroK, and tyrB to increase the supply of precursors; (3) employed a tyrA mutant strain to reduce carbon diversion and to result in non-growing cells; (4) enhanced the efflux of Phe by overexpressing yddG to shift equilibrium towards Phe synthesis and to release the feedback regulation in Phe synthesis. The mutants in PTS were firstly compared and a crr⁻ mutant was firstly screened. The mutant AroG15 was demonstrated to a thermostable mutant. The strains expressing yddG excreted Phe into the medium at higher rate and less intracellular Phe accumulated. By systems level engineering, an engineered Phe producer achieved 47.0 g/L Phe with a yield of 0.252 g/g which was the highest under the non-optimized fermentation condition.     

Preventive study in subjects at risk of fatal familial insomnia: Innovative approach to rare diseases

Abstract

The text describes a preventive clinical trial with drug treatment in a very rare neurodegenerative disease (Fatal familial Insomnia, FFI) designed with the help of individuals at genetic risk of developing the disease, asymptomatic carriers, who have agreed to be exposed over a 10-year period to doxycycline, an antibiotic with anti-prion activity. At least 10 carriers of the FFI mutation over 42 y old will be treated with doxycycline (100 mg/die) and the incidence of the disease will be compared to that of an historical dataset. For ethical reasons a randomized, double-blind, placebo-controlled trial was not feasible, however the study design and the statistical analysis ensure the scientific value of the results. This approach might represent an important breakthrough in terms of potential therapy and knowledge of rare diseases that could give some hopes to these neglected patients.     

Optimal cofactor swapping can increase the theoretical yield for chemical production in Escherichia coli and Saccharomyces cerevisiae

Maintaining cofactor balance is a critical function in microorganisms, but often the native cofactor balance does not match the needs of an engineered metabolic flux state. Here, an optimization procedure is utilized to identify optimal cofactor-specificity “swaps” for oxidoreductase enzymes utilizing NAD(H) or NADP(H) in the genome-scale metabolic models of Escherichia coli and Saccharomyces cerevisiae. The theoretical yields of all native carbon-containing molecules are considered, as well as theoretical yields of twelve heterologous production pathways in E. coli. Swapping the cofactor specificity of central metabolic enzymes (especially GAPD and ALCD2x) is shown to increase NADPH production and increase theoretical yields for native products in E. coli and yeast—including l-aspartate, l-lysine, l-isoleucine, l-proline, l-serine, and putrescine—and non-native products in E. coli—including 1,3-propanediol, 3-hydroxybutyrate, 3-hydroxypropanoate, 3-hydroxyvalerate, and styrene.     

Symmetrical aryl linked bis-iminothiazolidinones as new chemical entities for the inhibition of monoamine oxidases: Synthesis,in vitro biological evaluation and molecular modelling analysis

The multifactorial nature of Parkinson’s disease necessitates the development of new chemical entities with inherent ability to address key pathogenic processes. To this end, two series of new symmetrical 1,2- and 1,4-bis(2-aroyl/alkoylimino-5-(2-methoxy-2-oxoethylidene)-4-oxo-thiazolidin-3-yl)benzene derivatives (3a–g and 5a–e) were synthesized in good yields by the cyclization of 1,2- and 1,4-bis(N′-substituted thioureido)benzene intermediates with dimethyl acetylenedicarboxylate (DMAD) in methanol at ambient temperature. The bis-iminothiazolidinone compounds were investigated in vitro for their inhibition of monoamine oxidase (MAO-A & MAO-B) enzymes with the aim to identify new and distinct pharmacophores for the treatment of neurodegenerative disorders like Parkinson’s disease. Most of the designed compounds exhibited good inhibitory efficacy against monoamine oxidases. Compound 5a was identified as the most potent inhibitor of MAO-A depicting an IC₅₀ value of 0.001 μM, a 4-fold stronger inhibitory strength compared to standard inhibitor (clorgyline: IC₅₀ = 0.0045 μM). Molecular docking studies provided insights into enzyme-inhibitor interactions and a rationale for the observed inhibition towards monoamine oxidases.