Genome Evolution from Random Ligation of RNAs of Autocatalytic Sets

The evolutionary origin of the genome remains elusive. Here, I hypothesize that its first iteration, the protogenome, was a multi-ribozyme RNA. It evolved, likely within liposomes (the protocells) forming in dry-wet cycling environments, through the random fusion of ribozymes by a ligase and was amplified by a polymerase. The protogenome thereby linked, in one molecule, the information required to seed the protometabolism (a combination of RNA-based autocatalytic sets) in newly forming protocells. If this combination of autocatalytic sets was evolutionarily advantageous, the protogenome would have amplified in a population of multiplying protocells. It likely was a quasispecies with redundant information, e.g., multiple copies of one ribozyme. As such, new functionalities could evolve, including a genetic code. Once one or more components of the protometabolism were templated by the protogenome (e.g., when a ribozyme was replaced by a protein enzyme), and/or addiction modules evolved, the protometabolism became dependent on the protogenome. Along with increasing fidelity of the RNA polymerase, the protogenome could grow, e.g., by incorporating additional ribozyme domains. Finally, the protogenome could have evolved into a DNA genome with increased stability and storage capacity. I will provide suggestions for experiments to test some aspects of this hypothesis, such as evaluating the ability of ribozyme RNA polymerases to generate random ligation products and testing the catalytic activity of linked ribozyme domains.

Performance and Blood Metabolites of Growing Pigs Fed Paniculum Miliaceum Diet

During 30 days, the effects of millet grain regimen on performance indicators and blood metabolites in growing pigs were studied. A total of 40 Topigs pigs with similar age (81±3d) and weight (13.58±0.36 kg) were divided into two groups: control (C), based on the corn-triticale (25%)-soybean meal and experimental (M, where the millet replaces triticale). The production parameters and plasma samples were evaluated at the end of the experiment. Spotchem EZ SP-4430 analyzer from Arkray-Japan was used to assess the blood lipid, protein, enzyme, and mineral profiles. We noticed that the M diet maintains appropriate performance (33.22 vs. 31.30 final BW; 0.646 vs. 0.608 average daily gain; 1.39 vs. 1.29 average daily feed intake and, respectively 0.46 vs. 0.47 Gain: Feed ratio) and plasma metabolic profile with the C diet (P>0.05). In conclusion, the 25% millet added to the growing pigs' diet did not affect the performance indicators or body health.

Alcohol Acyltransferase Is Involved in the Biosynthesis of C6 Esters in Apricot (Prunus armeniaca L.) Fruit

Short-chain esters derived from fatty acid contribute to the characteristic flavor of apricot fruit, and the biosynthesis of these compounds in fruit is catalyzed by alcohol acyltransferase (AAT). In this work, we investigated the AAT gene family via genome-wide scanning, and three AAT loci were identified in different linkage groups (LGs), with PaAAT1 (PARG22907m01) in LG7, PaAAT2 (PARG15279m01) in LG4, and PaAAT3 (PARG22697m01) in LG6. Phylogenetic analysis showed that PaAAT1 belongs to clade 3, while PaAAT2 and PaAAT3 belong to clade 1 and clade 2, respectively. In contrast, the three AAT genes present different expression patterns. Only PaAAT1 exhibited distinct patterns of fruit-specific expression, and the expression of PaAAT1 sharply increased during fruit ripening, which is consistent with the abundance of C4–C6 esters such as (E)-2-hexenyl acetate and (Z)-3-hexenyl acetate. The transient overexpression of PaAAT1 in Katy (KT) apricot fruit resulted in a remarkable decrease in hexenol, (E)-2-hexenol, and (Z)-3-hexenol levels while significantly increasing the corresponding acetate production (p < 0.01). A substrate assay revealed that the PaAAT1 protein enzyme can produce hexenyl acetate, (E)-2-hexenyl acetate, and (Z)-3-hexenyl acetate when C6 alcohols are used as substrates for the reaction. Taken together, these results indicate that PaAAT1 plays a crucial role in the production of C6 esters in apricot fruit during ripening.

TAT for Enzyme/Protein Delivery to Restore or Destroy Cell Activity in Human Diseases

Much effort has been dedicated in the recent decades to find novel protein/enzyme-based therapies for human diseases, the major challenge of such therapies being the intracellular delivery and reaching sub-cellular organelles. One promising approach is the use of cell-penetrating peptides (CPPs) for delivering enzymes/proteins into cells. In this review, we describe the potential therapeutic usages of CPPs (mainly trans-activator of transcription protein, TAT) in enabling the uptake of biologically active proteins/enzymes needed in cases of protein/enzyme deficiency, concentrating on mitochondrial diseases and on the import of enzymes or peptides in order to destroy pathogenic cells, focusing on cancer cells.

PREDICTION OF FUNCTIONAL, STRUCTURAL AND STABILITY CHANGES IN PMM2 GENE ASSOCIATED WITH NEPHROTIC SYNDROME USING COMPUTATIONAL ANALYSIS

Objective: Nephrotic syndrome defines as a disorder with a group of symptoms like proteinuria, hypoalbuminemia, hyperlipidemia, and edema. PMM2 encodes phosphomannosemutase protein enzyme involved in the synthesis of N-glycan. Methods: Different Insilico analysis tools: SIFT, PolyPhen, PROVEAN, SNPandGO, MetaSNP, PhDSNP, MutPred, I-Mutant, STRUM, PROCHECK-Ramachandran, COACH and ConSurf, were used to check the effect of nsSNP on protein structure and function. Results: The genetic polymorphism in the PMM2 gene was retrieved from NCBI ClinVar and UniProtKB. Total 20 SNPs were predicted most significant and responsible for disease-causing and decrease protein stability. Conclusion: This study helps to discover disease-causing deleterious SNPs with different computational tools and gives information about potent SNPs.

Lipid vesicle fusion as a vehicle to study transmembrane protein interactions

Alzheimer's disease, among other neurologically degenerative diseases, has been linked to protein-enzyme interactions that originate within the transmembrane domain of a cell. The lipid environment that houses these interactions lends difficulty to studying intramembrane interactions, often making for time consuming data analysis. Lengthy data interpretation on top of the rate in which protein-enzyme interactions take place creates a need for a method to overcome these obstacles. The use of lipid vesicle fusion to apply a margin of control over the time frame of interaction combined with deep probing spectroscopic techniques can minimize the interference of the lipid environment. Deep ultraviolent resonance Raman (dUVRR) is a vibrational spectroscopic technique that probes along the protein backbone that allows for removal of lipid environmental interference through background subtraction. Lipid vesicle fusion has been demonstrated by mixing lipid vesicles comprised of oppositely charged head groups (cationic 1,2-diaurroyl-sn-glycero-3-phospho-(1-rac-glycerol) (DLPG)) and anionic 1,2-dilauroyl-sn-glycero-3-ethylphocholine (12:0 EPC) or 1,2-dimyristoyl-sn-glycero-3-ethylphocholine (14:0 EPC)) of equivalent or varying aliphatic tail length, up to a 2-carbon difference. The fluorescent dye, 8-aminonapthalene 1,3,6-trisulfonic acid (ANTS), paired with the quencher, p-xylene-bis-pyridiumbromide (DPX), are separately encased in either DLPG or 12:0 EPC/14:0 EPC, respectively, in aqueous solution, and evidence of lipid vesicle fusion is provided by monitoring fluorescence intensity of ANTS as the two solutions are mixed, resulting in the closing proximity of ANTS and DPX observed as a decrease in fluorescence intensity. Additional evidence is provided by dynamic light scattering (DLS) measurements of both independent vesicle solutions and their mixture showing an increase in hydrodynamic radius (Rh). In addition, cohesion of similarly sized lipids is demonstrated, as DLPG (12-carbon chain) fails to fuse with cationic lipids of chain length 16 carbons or longer. Circular dichroism (CD) is a spectroscopic technique that uses left and right-handed polarized light to obtain the overall secondary structure of proteins. Shown is the use of CD to probe the secondary structure and the changes incurred on PolyLA7 (PLA7), a model ?-helical peptide when placed in a transmembrane or hydrophobic environment, through a change in the lipid environment. PLA7 was inserted in DLPG lipid vesicles and then mixed in solution with lipid vesicles comprised of 14:0 EPC. CD spectra were obtained pre and post vesicle fusion, demonstrating the use of lipid fusion as a means to combine membrane embedded proteins of interest while still being able to observe changes that take place. Finally, we propose an on-demand lipid fusion system in which two separate lipid vesicles could be co-suspended in solution and then chemically or photonically induced to fuse. A titration was performed to obtain the pKa of a synthesized pH inducible cationic lipid (pHiCL). The pHiCL is a dipicolylamine with an attached 12-carbon aliphatic tail. The pHiCL was titrated while suspended in an aqueous environment and while inserted into a lipid vesicle comprised of DLPC, a net neutral lipid also with a 12-carbon length aliphatic tail. The pHiCL will be the first component of a two-part system in which a photoacid (PA) will be used to protonate the pHiCL in solution giving rise to cationic and anionic surfaced lipid vesicles causing vesicle fusion to occur.

A Tale of Two Bioconjugations: pH Controlled Divergent Reactivity of Protein A-Oxo Aldehydes in Competing A-Oxo-Mannich and Catalyst-Free Aldol Ligations

<div><div><div><p>Site-selective chemical methods for protein bioconjugation have revolutionised the fields of cell and chemical biology through the development of novel protein/enzyme probes bearing fluorescent, spectroscopic or even toxic cargos. Herein we report two new methods for the bioconjugation of a-oxo aldehyde handles within proteins using small molecule aniline and/or phenol probes. The ‘a-oxo-Mannich’ and ‘catalyst-free aldol’ ligations both compete for the electrophilic a-oxo aldehyde which displays pH divergent reactivity proceeding through the “Mannich” pathway at acidic pH to afford bifunctionalised bioconjugates, and the “catalyst-free aldol” pathway at neutral pH to afford monofunctionalised bioconjugates. We explore the substrate scope and utility of both these bioconjugations in the construction of neoglycoproteins, in the process formulating a mechanistic rationale for how both pathways intersect with each other at different reaction pH.</p></div></div></div>

A Tale of Two Bioconjugations: pH Controlled Divergent Reactivity of Protein A-Oxo Aldehydes in Competing A-Oxo-Mannich and Catalyst-Free Aldol Ligations

<div><div><div><p>Site-selective chemical methods for protein bioconjugation have revolutionised the fields of cell and chemical biology through the development of novel protein/enzyme probes bearing fluorescent, spectroscopic or even toxic cargos. Herein we report two new methods for the bioconjugation of a-oxo aldehyde handles within proteins using small molecule aniline and/or phenol probes. The ‘a-oxo-Mannich’ and ‘catalyst-free aldol’ ligations both compete for the electrophilic a-oxo aldehyde which displays pH divergent reactivity proceeding through the “Mannich” pathway at acidic pH to afford bifunctionalised bioconjugates, and the “catalyst-free aldol” pathway at neutral pH to afford monofunctionalised bioconjugates. We explore the substrate scope and utility of both these bioconjugations in the construction of neoglycoproteins, in the process formulating a mechanistic rationale for how both pathways intersect with each other at different reaction pH.</p></div></div></div>

Genome Evolution from Random Ligation of RNAs of Autocatalytic Sets

The evolutionary origin of the genome remains elusive. Here, I hypothesize that its first iteration, the protogenome, was a multi-ribozyme RNA. It evolved, likely within liposomes (the protocells) forming in dry-wet cycling environments, through the random fusion of ribozymes by a ligase and was amplified by a polymerase. The protogenome thereby linked, in one molecule, the information required to seed the protometabolism (a combination of RNA-based autocatalytic sets) in newly forming protocells. If this combination of autocatalytic sets was evolutionarily advantageous, the protogenome would have amplified in a population of multiplying protocells. It likely was a quasispecies with redundant information, e.g., multiple copies of one ribozyme. As such, new functionalities could evolve, including a genetic code. Once one or more components of the protometabolism were templated by the protogenome (e.g., when a ribozyme was replaced by a protein enzyme), and/or addiction modules evolved, the protometabolism became dependent on the protogenome. Along with increasing fidelity of the RNA polymerase, the protogenome could grow, e.g., by incorporating additional ribozyme domains. Finally, the protogenome could have evolved into a DNA genome with increased stability and storage capacity. I will provide suggestions for experiments to test some aspects of this hypothesis.