Simplified methodology for a modular and genetically expanded protein synthesis in cell-free systems

Genetic code expansion,which enables the site-specific incorporation of unnatural amino acids into proteins,has emerged as a new and powerful tool for protein engineering.Currently,it is mainly utilized inside living cells for a myriad of applications.However,the utilization of this technology in a cell-free,reconstituted platform has several advantages over living systems.The typical limitations to the employment of these systems are the laborious and complex nature of its preparation and utilization.Herein,we describe a simplified method for the preparation of this system from Escherichia coli cells,which is specifically adapted for the expression of the components needed for cell-free genetic code expansion.Besides,we propose and demonstrate a modular approach to its utilization.By this approach,it is possible to prepare and store different extracts,harboring various translational components,and mix and match them as needed for more than four years retaining its high efficiency.We demonstrate this with the simultaneous incorporation of two different unnatural amino acids into a reporter protein.Finally,we demonstrate the advantage of cell-free systems over living cells for the incorporation ofδ-thio-boc-lysine into ubiquitin by using the methanosarcina mazei wild-type pyrrolysyl tRNACUA and tRNA-synthetase pair,which could not be achieved in a living cell.

Targeting the overexpressed mitochondrial protein VDAC1 in a mouse model of Alzheimer’s disease protects against mitochondrial dysfunction and mitigates brain pathology

Background Alzheimer’s disease(AD)exhibits mitochondrial dysfunctions associated with dysregulated metabolism,brain inflammation,synaptic loss,and neuronal cell death.As a key protein serving as the mitochondrial gatekeeper,the voltage-dependent anion channel-1(VDAC1)that controls metabolism and Ca2+homeostasis is positioned at a convergence point for various cell survival and death signals.Here,we targeted VDAC1 with VBIT-4,a newly developed inhibitor of VDAC1 that prevents its pro-apoptotic activity,and mitochondria dysfunction.Methods To address the multiple pathways involved in AD,neuronal cultures and a 5×FAD mouse model of AD were treated with VBIT-4.We addressed multiple topics related to the disease and its molecular mechanisms using immunoblotting,immunofluorescence,q-RT-PCR,3-D structural analysis and several behavioral tests.Results In neuronal cultures,amyloid-beta(Aβ)-induced VDAC1 and p53 overexpression and apoptotic cell death were prevented by VBIT-4.Using an AD-like 5×FAD mouse model,we showed that VDAC1 was overexpressed in neurons surrounding Aβplaques,but not in astrocytes and microglia,and this was associated with neuronal cell death.VBIT-4 prevented the associated pathophysiological changes including neuronal cell death,neuroinflammation,and neuro-metabolic dysfunctions.VBIT-4 also switched astrocytes and microglia from being pro-inflammatory/neurotoxic to neuroprotective phenotype.Moreover,VBIT-4 prevented cognitive decline in the 5×FAD mice as evaluated using several behavioral assessments of cognitive function.Interestingly,VBIT-4 protected against AD pathology,with no significant change in phosphorylated Tau and only a slight decrease in Aβ-plaque load.Conclusions The study suggests that mitochondrial dysfunction with its gatekeeper VDAC1 is a promising target for AD therapeutic intervention,and VBIT-4 is a promising drug candidate for AD treatment.

Aromatic alcohols oxidation and hydrogen evolution over π-electron conjugated porous carbon nitride rods

Photocatalysis using polymeric carbon nitride(CN)materials is a constantly evolving field,where the variation of synthetic procedures allows the constant improvement of activity by tackling the intrinsic limitations of these materials(optical absorbance,specific surface area,charge migration,etc.).Amongst the possible photocatalytic reactions,the most popular application of CNs is the hydrogen evolution reaction(HER)from water.In this work,we design precisely-controlled carbon-doped porous CN rods with extended π-electron conjugation from supramolecular assemblies of melem and co-monomers,which partially substitute nitrogen for carbon atoms at the pyrimidine ring of the melem.Dense hydrogen bonds and good thermal stability of the melem-based supramolecular framework allow synthesizing a more ordered structure for improved charge migration;the control from the molecular level over the position of carbon-substituted nitrogen positions tailors the band alignment and photogenerated charge separation.The optimal photocatalyst shows an excellent HER rate(up to 10.16 mmol·h-1·g-1 under 100 W white light-emitting diode(LED)irradiation,with an apparent quantum efficiency of 20.0%at 405 nm,which is 23.2 times higher compared to a reference bulk CN).To fully harness the benefits of the developed metal-free CNs,selective oxidation reaction of aromatic alcohols is demonstrated with high conversion and selectivity.

Correction:Targeting the overexpressed mitochondrial protein VDAC1 in a mouse model of Alzheimer’s disease protects against mitochondrial dysfunction and mitigates brain pathology

Correction:Translational Neurodegeneration(2022)11:58 https://doi.org/10.1186/s40035-022-00329-7 Following publication of this article[1],the authors iden-tified an error in the family name of Alon Monsonego.The incorrect author name is:Alon Monsengo.The correct author name is:Alon Monsonego.The author group has been updated above and the orig-inal article[1]has been corrected.

Three-dimensional single particle tracking using 4π self-interference of temporally phase-shifted fluorescence

Single particle tracking in three dimensions is an indispensable tool for studying dynamic processes in various disciplines,including material sciences,physics,and biology,but often shows anisotropic three-dimensional spatial localization precision,which restricts the tracking precision,and/or a limited number of particles that can be tracked simultaneously over extended volumes.Here we developed an interferometric,three-dimensional fluorescence single particle tracking method based on conventional widefield excitation and temporal phase-shift interference of the emitted,high-aperture-angle,fluorescence wavefronts in a greatly simplified,free-running,triangle interferometer that enables tracking of multiple particles at the same time with<10-nm spatial localization precision in all three dimensions over extended volumes(~35×35×2μm^(3))at video rate(25 Hz).We applied our method to characterize the microenvironment of living cells and up to~40μm deep in soft materials.