Muscle is one of the most critical organs for mammals, which governs multiple movement and physiological functions. Circular RNA (circRNA) is a kind of novel endogenous RNA without 5'-Caps and 3'-poly(A) structures formed by pre-mRNA's back-splicing. RNA binding proteins (RBPs) control the production and degradation of circRNA, help nucleus-cytoplasm transport and locate circRNA, and regulate circRNA translation. Therefore, circRNAs and the chaperoned RBPs play critical roles in muscle growth, development, and disease progression. In this review, we systematically characterize the possible molecular mechanism of circRNA-protein interactions. Also, we summarize the latest researches on circRNA-protein interactions in muscle development and diseases. Besides, we provide several valid prediction methods and experimental verification approaches. Our review reveals the importance of circRNAs and their protein chaperones and provides a reference for further study in this field.

The cucumber mosaic virus (CMV) 2b protein is a suppressor of plant defenses and a pathogenicity determinant. Amongst the 2b protein’s host targets is the RNA silencing factor Argonaute 1 (AGO1), which it binds to and inhibits. In Arabidopsis thaliana, if 2b-induced inhibition of AGO1 is too efficient it induces reinforcement of antiviral silencing by AGO2, and triggers increased resistance against aphids, CMV’s insect vectors. These effects would be deleterious to CMV replication and transmission, respectively, but are moderated by the CMV 1a protein by sequestering sufficient 2b protein molecules into P-bodies to prevent excessive inhibition of AGO1. Mutant 2b protein variants were generated and red and green fluorescent protein fusions used to investigate subcellular colocalization with AGO1 and the 1a protein, and the effects of mutations on complex formation with the 1a protein and AGO1 were investigated using bimolecular fluorescence complementation and co-immunoprecipitation assays. Although we found that residues 56-60 influenced the 2b protein’s interactions with the 1a protein and AGO1, it appears unlikely that any single residue or sequence domain is solely responsible. In silico predictions of intrinsic disorder within the 2b protein secondary structure were supported by circular dichroism (CD) but not by nuclear magnetic resonance (NMR) spectroscopy. Intrinsic disorder provides a plausible model to explain the 2b protein’s ability to interact with AGO1, the 1a protein and other factors. However, the reasons for the conflicting conclusions provided by CD and NMR must first be resolved.

Congenital Pouch Colon (CPC) is a rare anorectal anomaly common to North Western India specifically Rajasthan. Despite efforts to understand the clinical genetic makeup of CPC, no attempt on identifying non-coding RNAs was done. We have earlier reported CPC's rare variants from whole exome sequencing across 18 affected samples in a total of 64 subjects. A Smith-Waterman algorithm was used to infer a couple of lncRNAs from WES samples of CPC with predictions from the Noncode database. Further screening and quantification using PCR, we ascertained interactions using Micro Scale Thermophoresis (MST). We report the role of lnc-EPB41-1-1 shown to be promiscuously interacting with KIF13A substantiating their role in regulation.

The complexity of RNA cannot be fully expressed with the canonical A, C, G, and U alphabet. To date, over 140 distinct chemical modifications to RNA have been discovered. RNA modifications can profoundly impact the cellular outcomes of messenger RNAs (mRNAs), transfer and ribosomal RNAs, and noncoding RNAs. Additionally, aberrant RNA modifications are associated with human disease. RNA modifications are a rising topic within the fields of biochemistry and molecular biology. This review aims to provide budding scientists with an appreciation for the significance of RNA modifications, alongside the skills required to identify and fluently discuss fundamental RNA-protein interactions. The Pumilio RNA-binding protein and YT521-B homology (YTH) family of modified RNA-binding proteins serve as examples to highlight the fundamental biochemical interactions that underlie the specific recognition of both unmodified and modified ribonucleotides, respectively.

A defining feature of a productive infection is the co-opting of host cell resources for viral replication. Despite their repertoire of molecular functions, viruses subvert host defenses to take control of cellular factors such as RNA binding proteins (RBPs). RBPs are involved in virtually all steps of mRNA life forming ribonucleoprotein complexes (mRNPs) in a highly ordered and regulated process to control RNA fate and stability in the cell. Thus, the hallmark of this viral takeover is to reshape RNA fate in the cell to modulate host gene expression and evade immune responses. Here we provide an extensive review of work in this area, particularly how the host-viral interplay influences RBP functions to modulate the host cell. Overall, in this review, we highlight the myriad of ways RBPs can regulate RNA stability in either a pro-viral or antiviral manner by gathering novel insights gained from research studies in this field.

Study of RNA-protein interactions and identification of RNA targets are among the key aspects of understanding the RNA biology. Currently, various methods are available to investigate these interactions, in particular, RNA pulldown assay. In the present paper, a method based on the HaloTag technology is presented that is called Halo-RPD (HaloTag RNA PullDown). The proposed protocol uses plants with stable fusion protein expression and the MagneBeads magnetic beads to capture RNA-protein complexes directly from the cytoplasmic lysate of transgenic A. thaliana plants. The key stages described in the paper are as follows: 1) preparation of the magnetic beads 2) tissue homogenization and collection of control samples 3) precipitation and wash of RNA-protein complexes; 4) evaluation of protein binding efficacy; 5) RNA isolation; 6) analysis of the obtained RNA. Recommendations for better NGS assay designs are provided.

Noncoding RNAs (ncRNAs) including circular RNAs (circRNAs) and N6-methyladenosine (m6A) have been shown to play a critical role in the development of various diseases including obesity and metabolic disorder-associated fatty liver disease (MAFLD). Obesity is a chronic disease considered by excessive fat accumulation in the body, which has been increased lately and is the foremost risk factor for the MAFLD. Causes of obesity may involve the interaction of genetic, behavioral, and social factors. The m6A RNA methylation might add a novel inspiration for understanding the development of obesity and MAFLD with post-transcriptional regulation of gene expression. In particular, circRNAs, microRNAs (miRNAs) and m6A might be implicated in the progression of MAFLD. Interestingly, m6A modification could modulate the translation, degradation, and other functions of ncRNAs. miRNAs/circRNAs could also modulate m6A modifications by affecting writers, erasers, and readers. In turn, ncRNAs could modulate the expression of m6A regulators in different ways. However, there is limited evidence on how these ncRNAs and m6A interact to affect the promotion of liver diseases. M6A has been appeared to occur in DNA, RNA and proteins, which may be associated with several biological properties. This study would provide a mechanistic understanding of the m6A modification and ncRNAs in the association on liver diseases, especially for MAFLD. Comprehension of the association between m6A modification and ncRNAs may contribute to the development of treatment tactics for MAFLD.

Circular RNAs are a special type of RNAs which recently attracted a lot of research interest in studying its formation and function. RNA binding proteins (RBPs) that bind circRNAs are important in these processes but are relatively less studied. CLIP-Seq technology has been invented and applied to profile RBP-RNA interactions on the genome-wide scale. While mRNAs are usually the focus of CLIP-Seq experiments, RBP-circRNA interactions could also be identified through specialized analysis of CLIP-Seq datasets. However, many technical difficulties are involved in this process, such as the usually short read length of CLIP-Seq reads. In this study, we created a pipeline called Clirc specialized for profiling circRNAs in CLIP-Seq data and analyzing the characteristics of RBP- circRNAs interactions. In conclusion, this is one of the first few studies to investigate circRNAs and their binding partners through repurposing CLIP-Seq datasets to our knowledge, and we hope our work will become a valuable resource for future studies into the biogenesis and function of circRNAs. Clirc software is available at https://github.com/Minzhe/Clirc

Three finger toxins (3FTX) are a group of peptides that affect multiple receptor types. One group of proteins affected by 3FTX are nicotinic acetylcholine receptors (nAChR). Structural information on how neurotoxins interact with nAChR is limited and are confined to a small group of neurotoxins. Therefore, in silico methods are valuable in understanding the interactions between 3FTX and different nAChR subtypes, but there are no established protocols to model 3FTX – nAChR interactions. We developed a homology modeling and protein docking protocol to address this issue and tested its success on three different systems. First, neurotoxin peptides co-crystallized with acetylcholine binding protein (AChBP) were re-docked to assess whether Rosetta protein – protein docking can reproduce the native poses. Second, experimental data on peptide binding to AChBP was used to test whether the docking protocol can qualitatively distinguish AChBP-binders from non-binders. Finally, we docked eight peptides with known α7 and muscle-type nAChR binding properties to test whether the protocol can explain the differential activities of the peptides at the two receptor subtypes. Overall, our docking protocol succeeded in predicting both qualitative and specific aspects of 3FTX binding to nAChR and shed light on some unknown aspects of 3FTX binding to different receptor subtypes.

Studies on Protein-Protein interactions (PPI) can be helpful for the annotation of unknown protein function and for the understanding of cellular processes, such as specific virulence mechanisms developed by bacterial pathogens. In that context, several methods have been extensively used in recent years for the characterization of Mycobacterium tuberculosis PPI to further decipher TB pathogenesis. This review aims at compiling the most striking results based on in vivo methods (yeast and bacterial two-hybrid systems, protein complementation assays) for the specific study of PPI in mycobacteria. Moreover, newly developed methods, such as in-cell native mass resonance and proximity-dependent biotinylation identification, will have a deep impact on future mycobacterial research, as they are able to perform dynamic (transient interactions) and integrative (multiprotein complexes) analyses.

Mitochondria are semiautonomous organelles, which are pivotal for the energy metabolism of eukaryotic cells. These organelles contain their own genetic system (mtDNA, mitogenome) that encodes to some structural RNAs and organellar proteins. The biogenesis and function of mitochondria is tightly regulated by nuclear-encoded protein cofactors. The mtDNAs of land plants are characteristically large, with a complex mode of gene organization and expression, in particularly at the post-transcriptional level. To become functional, the primary organellar transcripts undergo extensive maturation steps, which include endo- and exo-nucleolytic cleavage, RNA-base editing, and both ‘cis’- and ‘trans’-splicing events. These essential processing steps rely on the activities of a large set of nuclear-encoded factors. Among these, the RNA helicases serve as key players in organellar (mt-)RNA metabolism. This review summarizes the important roles played by these factors in regulating the highly dynamic processes of transcription, RNA processing, and translation within the mitochondria of land plants. We further discuss recent advancements in understanding how dysregulation of mitochondrial RNA helicases affects the organellar gene expression, respiratory functions, and hence the growth, development and physiology of land plants.

The Zika virus (ZIKV) is a mosquito-borne Flavivirus and can be transmitted through an infected mosquito bite or through human-to-human interaction by sexual activity, blood transfusion, breastfeeding or perinatal exposure. After the 2015-2016 outbreak in Brazil, a strong link between ZIKV infection and microcephaly emerged. ZIKV specifically targets human neural progenitor cells, suggesting that proteins encoded by ZIKV bind and inactivate host cell proteins leading to microcephaly. Here, we present a systematic annotation of interactions between human proteins and the seven non-structural ZIKV proteins corresponding to a Brazilian isolate. The interaction network was generated by combining tandem-affinity purification followed by mass spectrometry with yeast two-hybrid screens. We identified 150 human proteins, involved in distinct biological processes, as interactors to ZIKV non-structural proteins. Our interacting network is composed of proteins that have been previously associated with microcephaly in human genetic disorders and/or animal models. This study builds on previously published interacting networks of ZIKV and genes related to autosomal recessive primary microcephaly to generate a catalog of human cellular targets of ZIKV proteins implicated in processes related to microcephaly in humans. Collectively, this data can be used as a resource for future characterization of ZIKV infection biology and help create a basis for the discovery of drugs which may disrupt the interaction and reduce the health damage to the fetus.

In order to alter and adjust the shape of the membrane, cells harness various mechanisms of curvature generation. Many of these curvature generation mechanisms rely on the interactions between peripheral membrane proteins, integral membrane proteins, and lipids in the bilayer membrane. One of the challenges in modeling these processes is identifying the suitable constitutive relationships that describe the membrane free energy that includes protein distribution and curvature generation capability. Here, we review some of the commonly used continuum elastic membrane models that have been developed for this purpose and discuss their applications. Finally, we address some fundamental challenges that future theoretical methods need to overcome in order to push the boundaries of current model applications.

The current framework of evolutionary theory postulates that evolution relies on random mutations generating a diversity of phenotypes on which natural selection acts. This framework was established using a top-down approach as it originated from Darwinism, which is based on observations made on complex multicellular organisms, and then modified to fit a DNA-centric view. In this article, I argue that, based on a bottom-up approach starting from the physicochemical properties of nucleic and amino acid polymers, we should reject the facts that: i) natural selection plays a dominant role in evolution, and ii) the probability of mutations is independent of the generated phenotype. I will show that the adaptation of a phenotype to an environment does not correspond to organism fitness but rather corresponds to maintaining the genome stability and integrity. In a stable environment, the phenotype maintains the stability of its originating genome, and both (genome and phenotype) are reproduced identically. In an unstable environment (i.e., corresponding to variations in physicochemical parameters above a physiological range), the phenotype no longer maintains the stability of its originating genome but instead influences its variations. Indeed, environment- and cellular-dependent physicochemical parameters define the probability of mutations in terms of frequency, nature and location in a genome. Evolution is non-deterministic because it relies on probabilistic physicochemical rules, and evolution is driven by a bidirectional interplay between genome and phenotype, the phenotype ensuring the stability of the genotype in a cellular and environment physicochemical parameter-depending manner.

A model of the early RNA world is proposed. Nearly self-complementary sequences that could adopt double-stranded, smallhairpin-like (shRNA), structures would be selected for due to their greater hydrolytic stability. These would be phosphorylated attheir 5' ends. We suppose that dehydrating conditions arise (perhaps intermittently) in the early environment allowing amino acidsto condense with these RNA molecules. The resulting phosphate-amino acid anhydrides would play the role of early, charged,tRNAs. A crude genetic code could emerge owing to the greater resistance of some amino acid-shRNA pairings to hydrolysisrelative to others. Early on there is no division of labor between mRNAs and tRNAs; the same molecules perform both functions.But the first systems would have encoded little in the way of protein sequence information. Rather they would have served as catalysts for the random polymerization of amino acids. It is speculated that the selective advantage inhering in such systems lay intheir ability to supply raw materials for the formation of coacervates within which the various molecules essential to proto-lifecould be concentrated. This would greatly facilitate the necessary chemistries. The evolution of homochiral protein and RNA populations is discussed. An appealing feature of this model is its ability to explain the transition from phosphorylated amino acids to the 3' ester-linked aminoacyl-tRNAs employed by modern life.

Nanotechnology is a multidisciplinary science covering matters involving nanoscale level that is being developed for a great variety of applications. Nanomedicine is one of these attractive and challenging uses focused on the employment of nanomaterials in medical applications such as drug delivery. However, the uses of these nanometric systems requires specific parameters to establish the possible advantages and disadvantages in specific applications. This review presents the fundamental factors of nanoparticles and it´s microenvironment that must be considered to make an appropriate design for medical applications: (i) Interactions between nanoparticles and their biological environment, (ii) the interaction mechanisms, (iii) and the physicochemical properties of nanoparticles. On the other hand, the repercussions of the control, alteration and modification of these parameters in the final applications. Additionally, we here briefly report the implications of nanoparticles in nanomedicine and provide perspectives for some particular applications which still are challenged

The 2-oxoglutarate dehydrogenase complex (OGDHc) is a key enzyme in the TCA cycle and represents one of the major regulators of mitochondrial metabolism through NADH and reactive oxygen species levels. The OGDHc impacts cell metabolic and cell signaling pathways through the coupling of 2-oxoglutarate metabolism to gene transcription related to tumor cell proliferation and aging. DHTKD1 is a gene encoding 2-oxoadipate dehydrogenase (E1a), which functions in the L-lysine degradation pathway. The potentially damaging variants in DHTKD1 have been associated to the (neuro) pathogenesis of several diseases. Evidence was obtained for the formation of a hybrid complex between the OGDHc and E1a, suggesting a potential cross talk between the two metabolic pathways and raising fundamental questions about their assembly. Here we reviewed the recent findings and advances in understanding of protein-protein interactions in OGDHc and 2-oxoadipate dehydrogenase complex (OADHc), an understanding that will create a scaffold to help design approaches to mitigate the effects of diseases associated with dysfunction of the TCA cycle or lysine degradation. A combination of biochemical, biophysical and structural approaches such as chemical cross-linking MS and cryo-EM appears particularly promising to provide vital information for the assembly of 2-oxo acid dehydrogenase complexes, their function and regulation.

Influenza virus transcription is catalyzed by the viral RNA-polymerase (FluPol) through a cap-snatching activity. The snatching of the cap of cellular mRNA by FluPol is preceded by its binding to the flexible C-terminal domain (CTD) of the RPB1 subunit of RNA-polymerase II (Pol II). To better understand how FluPol brings the 3’-end of the genomic RNAs in close proximity to the host-derived primer, we hypothesized that FluPol may recognize additional Pol II subunits/domains to ensure cap-snatching. Using binary complementation assays between the Pol II and FluPol subunits and their structural domains, we revealed an interaction between the N-third domain of PB2 and RPB4. This interaction was confirmed by a co-immunoprecipitation assay and found to occur with the homologous domains of influenza B and C FluPols. Residues [1-72] of RPB4 were found critical in this interaction. Numerous punctual mutants generated at conserved positions between influenza A, B and C FluPols in the N-third domain of PB2 exhibited strong transcriptional activity defect. These results suggest that FluPol interacts with several domains/subunits of Pol II, the CTD to bind Pol II initiating host transcription and a second on RPB4 to locate FluPol at the proximity of the 5’-end of nascent host mRNA.

The non-structural protein (NS) and nucleoprotein (NP) of the severe fever with thrombocytopenia syndrome virus (SFTSV) encoded by the S segment are crucial for viral pathogenesis. They reside in viroplasm-like structures (VLS), but their interaction and their significance in viral propagation remain unclear. Here, we investigated the significance of the association between NS and NP during viral infection through in-silico and in-vitro analyses. Through in-silico analysis, three possible binding sites were predicted, at NSs1-32, NSs56-82, and NSs207-237. Rationally, three mutant NSs were developed by site-directed mutagenesis and tested for NP interaction by co-immunoprecipitation. NSsW61Y failed to interact with the nucleoprotein, which was substantiated by the conformational changes observed in the structural analyses. Additionally, molecular docking analysis corroborated that the NSW61Y mutant protein does not interact well compared to wild-type NS. Over-expression of wild-type NS in HeLa cells increased the SFTSV replication by five folds, but NSsW61Y exhibited 1.9-folds less viral replication than wild-type. We demonstrated that the W61Y alteration was implicated in the reduction of NS-NP interaction and viral replication. Thus, the present study identified a critical NS site, which could be targeted for development of therapeutic regimens against SFTSV.

This paper describes new horizons in the diversity and taxonomy of negev-like viruses encoding the membrane-embedded SP24 protein. First, our data extend the known host range of SP24-encoding negev-like viruses to include brown algae, fungi, green plants, the phylum Entoprocta, the phylum Mollusca, and vertebrates. Second, our phylogenetic analysis suggests that the evolution of the SP24 gene family may have involved frequent events of inter-order virus genome shuffling. Third, the identification of 2-3 copies of SP24 protein genes in some virus RNAs shows that virus genomes may have acquired additional SP24 genes during the evolutionary process due to duplications or new acquisition steps. Forth, the broad host specificity of some SP24-encoded viruses may be related to an important adaptive role of SP24. Fifth, insect and nematode genomes may acquire viral SP24 genes by putative horizontal transfer from negev-like viruses known to infect species of both taxa.

The outbreak of COVID-19, the disease caused by SARS-CoV-2, continues to affect millions of people around the world. The absence of a globally distributed effective treatment makes the exploration of new mechanisms of action a key step to address this situation. Stabilization of non-native Protein-Protein Interactions (PPIs) of the nucleocapsid protein of MERS-CoV has been reported as a valid strategy to inhibit viral replication. In this study, the applicability of this unexplored mechanism of action against SARS-CoV-2 is analyzed. During our research, we were able to find three inducible interfaces of SARS-CoV-2 N protein NTD, compare them to the previously reported MERS-CoV stabilized dimers, and identify those residues that are responsible for their formation. A drug discovery protocol implemented consisting of docking, molecular dynamics and MM-GBSA enabled us to find several compounds that might be able to exploit this mechanism of action. In addition, a common catechin skeleton was found among many of these molecules, which might be useful for further drug design. We consider that our findings could motivate future research in the fields of drug discovery and design towards the exploitation of this previously unexplored mechanism of action against COVID-19.

Ribosomal proteins (r-proteins) are abundant, highly conserved, and multifaceted cellular proteins in all domains of life. Most r-proteins have RNA-binding properties and are able to form protein-protein contacts. Bacterial r-proteins govern the co-transcriptional rRNA folding during ribosome assembly and participate in formation of the ribosome functional sites such as the mRNA binding site, tRNA binding sites, the peptidyl- transferase center, and the protein exit tunnel. In addition to their primary role in a cell as integral components of the protein synthesis machinery, many of r-proteins can function beyond the ribosome (the phenomenon known as moonlighting), acting either as individual regulatory proteins or in complexes with various cellular components. Extraribosomal activities of r-proteins have been intensively studied over decades (reviewed in [1]). The past decade brought significant advancement in our knowledge of r-protein functions due to expanding studies on ribosomes and gene expression mechanisms not only in model bacteria like Escherichia coli or Bacillus subtilis, but also in previously little-explored bacterial species from various phyla. This review is aimed to update the information on multiple functions of r-proteins in bacteria.

Kiwifruit is a perennial horticultural crop species with high nutritional and economic value. However, various pathogens stress brings a serious threat to kiwifruit. Pentatricopeptide repeat proteins (PPRs) are characterized by tandem repetitions of 35 amino acid motifs, and have been found to function in plant RNA editing. However, the roles of PPRs in plant development and disease resistance remain unclear. In this study, we performed a genome-wide identification and characterization of PPR gene family in two kiwifruit species (Actinidia chinensis (Ach), and Actinidia eriantha (Ace)) with markedly different disease resistance. A total of 497 and 499 PPRs were identified in Ach and Ace, respectively. All the kiwifruit PPRs could be phylogenetically divided into four subfamilies. There were about 40.68% PPRs predicted to be localized to mitochondria or chloroplasts. Synteny analysis showed that the expansion of kiwifruit PPRs mainly came from segmental duplication. Based on RNA-seq data from the fruit during 12 periods of development and maturity, weighted correlation network analysis suggested that two PPRs Actinidia20495.t1 and Actinidia15159.t1 were involved in the fruit development and maturation. In addition, we observed different gene expression of PPRs and chloroplast RNA editing profiles between resistant and susceptible kiwifruits after pathogen infection, indicating the roles of PPRs in stress response by modulating the editing extend of mRNA. The differentially expressed upstream transcription factors of PPRs were further identified, they may regulate resistance adaption by modulating the PPRs expression. These results provide a solid foundation for further analyses of the functions and molecular evolution of PPRs, in particular, for clarifying the resistance mechanisms in kiwifruits and breeding new cultivars with high resistance.

The provenance of the aminoacyl-tRNA synthetases (aaRS) poses challenging questions because of their role in the emergence and evolution of genetic coding. We investigate evidence about their ancestry from curated structure-based multiple sequence alignments of a structurally invariant “scaffold” shared by all 10 canonical Class I aaRS. Three uncorrelated phylogenetic metrics—residue-by-residue conservation, its variance, and row-by-row cladistic congruence—imply that the Class I scaffold is a mosaic assembled from distinct, successive genetic sources. These data are especially significant in light of: (i) experimental fragmentations of the Class I scaffold into three partitions that retain catalytic activities in proportion to their length; and (ii) evidence that two of these partitions arose from an ancestral Class I aaRS gene encoding a Class II ancestor in frame on the opposite strand. Phylogenetic metrics of different modules vary in accordance with their presumed functionality. A 46-residue Class I “protozyme” roots the Class I molecular tree prior to the adaptive radiation of the Rossmann dinucleotide binding fold that refined substrate discrimination. Such rooting is consistent with near simultaneous emergence of genetic coding and the origin of the proteome, resolving a conundrum posed by previous inferences that Class I aaRS evolved long after the genetic code had been implemented in an RNA world. Further, pinpointing discontinuous enhancements of aaRS fidelity establishes a timeline for the growth of coding from a binary amino acid alphabet.

The advancement of precision medicine critically depends on the robustness and specificity of the carriers used for the targeted delivery of effector molecules in the human body. Numerous nanocarriers have been explored in vivo, to ensure the precise delivery of molecular cargos via tissue-specific targeting, including the endocrine part of the pancreas, thyroid, and adrenal glands. However, even after reaching the target organ, the cargo-carrying vehicle needs to enter the cell and then escape from lysosomal destruction. Most of artificial nanocarriers suffer from intrinsic limitations that either prevent them from completing the specific delivery of the cargo. In this respect, extracellular vesicles (EVs) seem to be the natural tool for payload delivery due to their versatility and low toxicity. However, EV-mediated delivery is not selective and usually short-ranged. By inserting the viral membrane fusion proteins into exosomes, it is possible to increase the efficiency of membrane recognition and also ease the process of membrane fusion. This review describes the molecular details of the viral-assisted interaction between the target cell and extracellular vesicles. We also discuss the question of the usability of viral fusion proteins in developing extracellular vesicle-based nanocarriers with higher efficacy of payload delivery. Finally, this review specifically highlights the role of Gag and RNA binding proteins in RNA sorting into extracellular vesicles.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease (CHD) affecting 1 in 5,000 newborns. We constructed the interactome of 74 HLHS-associated genes identified from a large-scale mouse mutagenesis screen, augmenting it with 408 novel protein-protein interactions (PPIs) using our High-precision Protein-Protein Interaction Prediction (HiPPIP) model. The interactome is available on a webserver with advanced search capabilities (http://severus.dbmi.pitt.edu/wiki-HLHS). 364 genes including 73 novel interactors were differentially regulated in tissues/iPSC-derived cardiomyocytes of HLHS patients. Novel PPIs facilitated the identification of TOR signaling and endoplasmic reticulum stress modules. 60.5% of the interactome consisted of housekeeping genes that may harbor large-effect mutations and drive HLHS etiology but show limited transmission. Network proximity of diabetes, Alzheimer’s disease, and liver carcinoma-associated genes to HLHS genes suggested a mechanistic basis for their comorbidity with HLHS. Interactome genes showed tissue-specificity for sites of extracardiac anomalies (placenta, liver and brain). The HLHS interactome shared significant overlaps with the interactomes of ciliopathy and microcephaly-associated genes, with the shared genes respectively enriched for genes involved in intellectual disability and/or developmental delay, and neuronal death pathways. This supported the increased burden of ciliopathy variants and prevalence of neurological abnormalities observed among HLHS patients with developmental delay and microcephaly respectively.

In recent years, the role of non-coding RNAs (ncRNAs) in regulating cell physiology has begun to be better understood. Recent discoveries in viral molecular biology have revealed that such cellular functions are disturbed during viral infections mainly due to host cell ncRNAs, cellular factors, and virus-derived ncRNAs. Apart from the interplay between those molecules, other interactions derive from the specific folding of RNA virus genomes. These fulfill canonical regulation functions such as replication, translation, and viral packaging. In some cases, folds serve as precursors of small viral RNAs whose biogenesis is not yet clearly understood. Since ncRNAs and RNA viral genomes modulate complex molecular and cellular processes in viral infections, a new taxonomy is being proposed here overarching three main categories, considering the current information about ncRNA interactions in some well-known viral infections. The first category shows examples of host ncRNAs associated with the trigger of the immune response under viral infections. The second category describes interactions between the virus and host ncRNAs. The last category shows how the shape of the RNA viral genome is essential in processing RNAs derived from viruses. Finally, we introduce evidence of how these three categories can also work as a framework in order to organize known interactions of ncRNAs and cellular factors under DENV infection. This new taxonomy of interactions provides a comprehensive framework for organizing the ncRNA regulatory roles in the context of viral interactions and an RNA world.

Silencing of the fragile X mental retardation 1 (FMR1) gene and consequently lack of synthesis of FMR protein (FMRP) are associated with fragile X syndrome, which is one of the most prevalent inherited intellectual disabilities. FMRP is a multifunctional protein involved in many cellular functions in nearly all subcellular compartments under normal conditions and under conditions of cellular stress in both neuronal and non-neuronal cell types. This is achieved through its trafficking signals, nuclear localization signal (NLS), nuclear export signal (NES), and nucleolar localization signal (NoLS), as well as its RNA and protein binding domains, and is modulated by various post-translational modifications such as phosphorylation, ubiquitination, sumoylation, and methylation. This review summarizes recent advances in understanding the interaction networks of FMRP with a special focus on FMRP stress-related functions, including stress granule formation, mitochondrion and endoplasmic reticulum plasticity, ribosome biogenesis, cell cycle control, and DNA damage response.

Phenotypes are driven by regulated gene expression, which in turn are mediated by complex interactions between diverse biological molecules. Protein-DNA interactions such as histone and transcription factor binding are well studied, along with RNA-RNA interactions in short RNA silencing of genes. In contrast, lncRNA-protein interaction (LPI) mechanisms are comparatively unknown, likely driven by the difficulties in studying LPI. However, LPI are emerging as key interactions in epigenetic mechanism, playing a role in development and disease. Their importance is further highlighted by their conservation across kingdoms. Hence, interest in LPI research is increasing. We therefore review the current state of the art in lncRNA-protein interactions. We specifically surveyed recent computational methods and databases which researchers can exploit for LPI investigation. We discovered that algorithm development is heavily reliant on a few generic databases containing curated LPI information. We show that early methods predict LPI using molecular docking, have limited scope and are slow, creating a data processing bottleneck. Recently, machine learning has become the strategy of choice in LPI prediction, likely due to the rapid growth in machine learning infrastructure and expertise. While many of these methods have notable limitations, machine learning is expected to be the basis of modern LPI prediction algorithms.

The appearance of bubbles and foam can influence the likeability of a wine even before its consumption. Since foams are essential to visual and taste attributes of sparkling wines, it is of great importance to understand which compounds affect bubbles and foam characteristics. The aim of this work was to investigate the effect of interactions among proteins, amino acids, and phenols on the characteristics of foam in sparkling wines by using synchronous fluorescence spectroscopy techniques. Results has shown that several compounds present in sparkling wines influence foam quality differently, and importantly, highlighted how the interaction of those compounds might result in different effects on foam parameters. Amongst the results, mannoproteins were found to be most likely to promote foam and collar stability, while phenols were likely to increase the ratio of small bubbles and collar height in the foam matrix. In summary, this work contributes to a better understanding of the effect of wine compounds on foam quality as well as the effect of the interactions between those compounds.

Begomoviruses, which belong to the Geminiviridae family, are intracellular parasites transmitted by whiteflies to dicotyledonous plants, significantly damaging agronomically relevant crops. These nucleus-replicating DNA viruses need to move intracellularly from the nucleus to the cytoplasm and then, like other plant viruses, spread systemically throughout the plant to cause dis-ease. The transport proteins of begomoviruses play a crucial role in recruiting host components for the movement of viral DNA within and between cells while exhibiting functions that suppress the host's immune defense. Pioneering studies on species of the Begomovirus genus have identified specific viral transport proteins involved in intracellular transport, cell-to-cell movement, and systemic spread. Recent research has primarily focused on viral movement proteins and their interaction with the cellular host transport machinery, significantly expanding our understanding of viral infection pathways. This review focuses on three main aspects: (i) the role of viral transport proteins, specifically movement proteins (MPs) and nuclear shuttle proteins (NSPs), (ii) their ability to recruit host factors for intra- and intercellular viral movement, and (iii) suppress antiviral immunity, with a particular emphasis on bipartite begomoviral movement proteins.

Favipiravir is a broad-spectrum inhibitor of viral RNA-dependent RNA polymerase (RdRp) currently being used to manage COVID-19 in several countries. By acting as a substrate for RdRp, favipiravir gets incorporated into the nascent viral RNA and prevents strand extension. A high mutation rate of SARS-CoV-2 RdRp may facilitate antigenic drift as an answer to the host immune response, thereby generating resistance of virus to favipiravir. Therefore, it is extremely crucial to predict potential mutational sites in the RdRp and the emergence of structural modifications contributing to drug resistance. Here, we used high-throughput interface-based protein design to generate >100,000 designs and identify mutation hotspot residues in the favipiravir-binding site of RdRp. Several mutants had lower binding affinities to favipiravir, out of which hotspot residues with a high propensity to undergo positive selection were identified. The results showed that the designs retained an average of 97 to 98% sequence identity, suggesting that SARS-CoV-2 can develop favipiravir resistance with just a few mutations. Notably, we observed that out of 134 mutations predicted designs, 63 specific mutations were already present in the CoV-GLUE database, thus attaining ~47% correlation match with the clinical sequencing data. The findings improve our understanding of the potential signatures of adaptation in SARS-CoV-2 against favipiravir and management of COVID-19. Furthermore, they can help develop exhaustive strategies for robust antiviral design and discovery.

Climate change and globalization have raised the risk of vector-borne disease (VBD) introduction and spread in various European nations in recent years. In Italy, viruses carried by tropical vectors have been shown to cause viral encephalitis, one of the symptoms of arbovirosis, a spectrum of viral disorders spread by arthropods such as mosquitoes and ticks. Arbovirosis are currently causing alarm and attention, and the World Health Organization (WHO) has released recommendations to adopt essential measures, particularly during the hot season, to restrict the spreading of the infectious agents among breeding stocks. In this scenario, rapid analysis systems are required, because they can quickly provide information on potential virus-host interactions, the evolution of the infection, and the onset of disabling clinical symptoms, or serious illnesses. Such systems include bioinformatics approaches integrated with molecular evaluation. Viruses have co-evolved different strategies to transcribe their own genetic material, by changing the host's transcriptional machinery, even in short periods of time. The introduction of genetic alterations, particularly in RNA viruses, results in a continuous adaptive fight against the host's immune system. We suggest an in silico pipeline method to unravel viral sequences that may interact with host RNA binding proteins (RBPs), which play important roles in RNA metabolism and its several related biological processes. Indeed, viral RNA sequences, able to bind host RBPs may compete with cellular RNAs, altering important metabolic processes. Our findings suggest that the proposed in silico approach, could be a useful and promising tool to investigate the complex and multiform clinical manifestations of viral encephalitis, and possibly identify altered metabolic pathways as targets of pharmacological treatments and innovative therapeutic protocols.

Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) has claimed nearly 180,000 lives and continues to spread. There are currently no approved medications or vaccines for this new coronavirus. Studies have shown that the positive RNA genome of SARS-CoV-2 contains unique features, including a 12-base sequence inserted between the two subunits of viral receptor protein Spike. This inserted sequence facilitates the cleavage of Spike by the cellular proteases Furin and TMPRSS2, leading to the fusion of virus and host cell membranes. Current studies are mostly focused on the SARS-CoV-2 Spike protein and its interacting cellular proteins ACE2, Furin, and TMPRSS2. RNA structural studies are limited and little is known about the potential impact of the 12-base sequence insert on the secondary structure of SARS-CoV-2 genomic RNA and/or its transcripts. Here, by using local and global RNA secondary structure predictions, we show that the novel 12-base insert of SARS-CoV-2 genome likely induces a major RNA secondary structure change.

COVID19 is a current pandemic disease due to the novel coronavirus SARS-CoV-2. The scientific community mounted a strong response by accelerating research and innovation, and rapidly setting the basis to the understanding of molecular determinants of the disease for the development of targeted therapeutic interventions. The replication of the viral genome within the infected cells is a key step of SARS-CoV2 life cycle. It is a complex process involving the action of several viral and host proteins in order to perform RNA polymerization, proofreading and final capping. This review provides an update of structural and functional data on key actors of the replicatory machinery of SARS-CoV-2, filling the gaps in the current availability of structural data using homology modelling. Moreover, learning from similar viruses, we collect literature data to reconstruct the pattern of interactions among protein actors of the SARS-CoV-2 RNA polymerase machinery. In this pattern, an important role is played by co-factors, like Nsp8 and Nsp10, not only as allosteric activators but also as molecular connectors holding the entire machinery together to enhance the efficiency of RNA replication.

Endo-reticular stress induces the unfolded protein response including a highly conserved set of genes crucial for cell survival against a variety of onslaughts. Among the activated stress response genes is Ire1, which undergoes auto-phosphorylation and acquires a regulated Ire1-dependent mRNA decay activity. Ire1P non-canonically splices the mRNA for Xbp1 in the cytoplasm. This spliced Xbp1 serves as mRNA encoding a transcription factor for unfolded protein response genes. Meanwhile the mRNA decay function of Ire1P degrades other cellular mRNAs and can cause changes to the translation machinery by altering regulators in a cell specific manner. Naïve splenic B cells differentiate into Antibody Secreting Cells and activate Ire1 phosphorylation early on after LPS stimulation, within 18 hrs. When Ire1 is activated in B cells, in addition to Xbp1 splicing, there are large-scale changes in mRNA; inhibition of the mRNA degradation function of Ire1 both reduces the number and changes the type of genes involved in altered splicing patterns, including factors for snRNA transcription. Some of the splicing changes seen at 18 hrs after LPS persist into the late stages of antibody secretion, up to 72 hrs, while others are supplanted by new splicing changes introduced by the induction of ELL2, a transcription elongation factor. ELL2 changes mRNA processing patterns and is necessary for Immunoglobulin secretion. RNA splicing patterns in antibody secreting cells are thus shaped by endo-reticular stress, ELL2 induction, and are associated with changes in the levels of snRNAs.

Background: Recent developments in our understanding of the interactions between long non-coding RNA (lncRNA) and cellular components have improved treatment approaches for various human diseases including cancer, vascular diseases, and neurological diseases. Although investigation of specific lncRNAs revealed their role in the metabolism of cellular RNA, our understanding of their contribution to post-transcriptional regulation is relatively limited. In this study, we explore the role of lncRNAs in modulating alternative splicing and their impact on downstream protein-RNA interaction networks. Results: Analysis of alternative splicing events across 39 lncRNA wildtype and knockout RNA-sequencing datasets from three human cell lines: HeLa (Cervical Cancer), K562 (Myeloid Leukemia), and U87 (Glioblastoma), resulted in high confidence (fdr < 0.01) identification of 4432 skipped exon events and 2474 retained intron events, implicating 759 genes to be impacted at post-transcriptional level due to the loss of lncRNAs. We observed that a majority of the alternatively spliced genes in a lncRNA knockout were specific to the cell type, in agreement with the finding that genes affected by alternative splicing also displayed enriched functions in a cell type specific manner. To understand the mechanism behind this cell-type specific alternative splicing patterns, we analyzed RNA binding protein (RBP)-RNA interaction profiles across the spliced regions. Conclusions: Despite limited RBP binding data across cell lines, alternatively spliced events detected in lncRNA perturbation experiments were associated with RBPs binding in proximal intron-exon junctions, in a cell type specific manner. The cellular functions affected by alternative splicing were also affected in a cell type specific manner. Based on the RBP binding profiles in HeLa and K562 cells, we hypothesize that several lncRNAs are likely to exhibit a sponge effect in disease contexts, resulting in the functional disruption of RBPs due to altered titration of the RBPs from their target loci. We propose that such lncRNA sponges can extensively rewire the post-transcriptional gene regulatory networks by altering the protein-RNA interaction landscape in a cell-type specific manner.

Legume-rhizobial symbiosis leads to to the formation of root nodules, where rhizobia reside and develop into bacteroids to reduce nitrogen into ammonium for plant growth, which leaves an opening question as how plant immunity is attenuated in nodules in the presence of large number of foreign bacteria. In Medicago truncatula, mutation in NAD1 (Nodules with Activated Defense 1) only produces necrotic nodules with overactivated immunity, indicating NAD1 is an indispensable component required for suppressing immunity in nodules. In this study, a dual RNA-seq transcriptomic technology was performed to extensively analyze gene expressions in nad1-1 mutant nodules. We indenifited 89 DEGs in symbiotic nitrogen fixation and 89 DEGs in immunity in Medicago truncatula at 10 dpi. Simultaneously, we indenifited 27 DEGs in fix and nif genes in Sinorhizobium meliloti. Then we identified 56 DEGs related to stress, including ROS, NO, and NCR, most of which were upregulated in Sinorhizobium meliloti. Our analyses of nitrogen fixation-defective plant nad1-1 mutants with overactivated defense indicate that host use plant immunity to control massive bacterial colonization during early and late symbiotic stages. Our findings provide insight into the function of NAD1 in improvement or inhibition of plant immune response to maintain symbiosis during rhizobial endosymbiosis.

The RNA-binding protein Human antigen R (HuR) regulates stability, translation, and nucleus-to-cytoplasm shuttling of its target mRNAs. The protein has been progressively recognized as a relevant therapeutic target for several pathologies like cancer, neurodegeneration, as well as inflammation. Inhibitors of mRNA binding to HuR might thus be beneficial against a variety of diseases. Here we present the rational identification of structurally novel HuR inhibitors. In particular, by combining chemoinformatics approaches, high throughput virtual screening and RNA–protein pull-down assays, we show that the 4-(2-(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)hydrazineyl)benzoate ligand exhibits dose-dependent HuR inhibition in binding experiments. Importantly, the chemical scaffold is new with respect to the so-far known HuR inhibitors, opening up a new avenue for the design of pharmaceutical agents targeting this important protein.

Our health requires continual protein synthesis for maintaining and repairing tissues. For protein synthesis to function, all the essential (indispensable) amino acids (IAA) that must be available in the diet, along with those AAs that the cells can synthesize, the dispensable amino acids. Here we review studies that have shown the location of the detector for IAA deficiency in the brain, specifically for recognition of IAA deficient diets (IAAD diets) in the anterior piriform cortex (APC), with subsequent responses in downstream brain areas. The APC is highly excitable, uniquely suited to serve as an alarm for reductions in IAAs. With a balanced diet, these neurons are kept from over-excitation by GABAergic inhibitory neurons. Because several transporters and receptors on the GABAergic neurons have rapid turnover times, they rely on intact protein synthesis to function. When an IAA is missing, its unique tRNA cannot be charged. This activates the enzyme General Control Nonderepressible 2 (GCN2) that is important in the initiation phase of protein synthesis. Without the inhibitory control supplied by GABAergic neurons, excitation in the circuitry is free to signal an urgent alarm. Studies in rodents have shown rapid recognition of IAA deficiency by quick rejection of the IAAD diet.

Cells form a bipolar spindle during mitosis to ensure accurate chromosome segregation. Proper spindle architecture is established by a set of kinesin motors and microtubule-associated proteins. In most eukaryotes, kinesin-5 motors are essential for this process, and genetic or chemical inhibition of their activity leads to the emergence of monopolar spindles and cell death. However, these deficiencies can be rescued by simultaneous inactivation of kinesin-14 motors, as they counteract kinesin-5. We conducted detailed genetic analyses in fission yeast to understand the mechanisms driving spindle assembly in the absence of kinesin-5. Here we show that deletion of the nrp1 gene, which encodes a putative RNA-binding protein with unknown function, can rescue temperature sensitivity caused by cut7-22, a fission yeast kinesin-5 mutant. Interestingly, kinesin-14/Klp2 levels on the spindles in the cut7 mutants were significantly reduced by the nrp1 deletion, although the total levels of Klp2 and the stability of spindle microtubules remained unaffected. Moreover, RNA-binding motifs of Nrp1 are essential for its cytoplasmic localization and function. We have also found that a portion of Nrp1 is spatially and functionally sequestered by chaperone-based protein aggregates upon mild heat stress and limits cell division at high temperatures. We propose that Nrp1 might be involved in post-transcriptional regulation through its RNA-binding ability to promote the loading of Klp2 on the spindle microtubules.

Single molecule de novo protein sequencing based on the 'superspecificity' of amino-acyl tRNA synthetases (aaRS) is proposed. An unfolded protein molecule is threaded through a nanopore in an electrolytic cell (e-cell) to expose the terminal residue in the e-cell's trans chamber. After the residue is cleaved with an exopeptidase, a set of tRNAs, their aaRSs, and ATP are added to trans. An aaRS charges a cognate tRNA molecule with the residue. The charged tRNA (along with the other reactants) is transferred to an extended e-cell with N (20 ≤ N ≤ 61) pores in N individual cis chambers and a single trans chamber. Each pore holds an RNA molecule ending in a unique codon that is exposed in trans. The charged tRNA's anticodon base-pairs with the terminal codon of an RNA. If tRNAs and residues are fluorescently tagged with two different colors, the residue can be identified from the observed position of the resulting color pair. As charging is 'superspecific' identification is unambiguous. The protein molecule in the first e-cell is advanced by one residue and the process repeated. In this approach there is no need for precise pore current or optical intensity measurements. Potential implementation issues are discussed. Other possibilities, including one in which the terminal residue is cleaved after charging, are also examined.

Under the oxidative stress condition, the small RNA (sRNA) Oxys that acts as essential post-transcriptional regulators of gene expression is produced and plays a regulatory function with the assistance of the RNA chaperone Hfq protein. Interestingly, experimental studies found that the N48A mutation of Hfq protein could enhance the binding affinity with OxyS while resulting in defection of gene regulation. But, how the Hfq protein interacts with sRNA Oxys and the origin of the stronger affinity of N48A mutation are both unclear. In this paper, molecular dynamics (MD) simulations were performed on the complex structure of Hfq and OxyS to explore their binding mechanism. The molecular mechanics generalized Born surface area (MM/GBSA) and interaction entropy (IE) method were combined to calculate the binding free energy between Hfq and OxyS sRNA, and the computational result is in excellent correlation with the experimental result. Per-residue decomposition of the binding free energy revealed that the enhanced binding ability of the N48A mutation mainly comes from the increased van der Waals interactions (vdW). This research explores the binding mechanism between Oxys and chaperone protein Hfq, and revealed the origin of the strong binding affinity of N48A mutation. The results provided important insights on the mechanism of gene expression regulation affected by protein mutations.

Yeast two-hybrid approaches, which are based on fusion proteins that must co-localise to the nucleus to reconstitute the transcriptional activity of GAL4 have greatly contributed to understand the nitrogen interaction network of cyanobacteria, whose main hubs are the trimeric PII and the monomeric PipX regulators. The bacterial two-hybrid system, based on the reconstitution in the E. coli cytoplasm of the adenylate cyclase of Bordetella pertussis, should provide a relatively faster and presumably more physiological assay for cyanobacterial proteins than the yeast system. Here we use the bacterial two-hybrid system to gain additional insights into the cyanobacterial PipX interaction network while simultaneously assessing the advantages and limitations of the two most popular two-hybrid systems. Comprehensive mutational analysis of PipX and bacterial two-hybrid assays were performed to compare outcomes between yeast and bacterial systems. We detected interactions previously recorded in the yeast two-hybrid system as negative as well as a “false positive”, the self-interaction of PipX, which is rather an indirect interaction dependent on PII homologs from the E. coli host, a result confirmed by Western analysis with relevant PipX variants. This is, to our knowledge, the first report of the molecular basis of a false positive in the bacterial two-hybrid system.

Abstract: Due to their rapid evolution and their impact on health care, be-ta-lactamases, protein degrading beta-lactam antibiotics, are used as generic model of protein evolution. Therefore, we investigated the mutation effects in two distant beta-lactamases TEM-1 and CTX-M-15. Interestingly we found a site with a complex pattern of genetic interactions. Mutation G251W in TEM-1 is in-activating the protein’s function just as the reciprocal mutation W251G in CTXM-15. Phylogenetic analysis revealed that mutation G has been entrenched in TEM-1 background: while rarely observed throughout the phylogeny it is es-sential in TEM-1. Using a rescue experiment in TEM-1 G251W mutant, we could identify sites that alleviates the deviation from G to W. While few of these muta-tions could potentially involve local interactions, most of them were found on distant residues in the 3D structure. Many well-known mutations having an impact on protein stability, such as M182T, were recovered. Our results there-fore suggest that entrenchment of an amino acid may rely on diffuse interactions among multiple sites with a major impact on protein stability.

This research focussed on utilisation of salmon protein and lipid to manipulate pasta glycaemic index and protein digestibility. Salmon fish (Oncorhynchus tschawytscha) powder (SFP) supplemented pasta flour at the from 5% to 20% (w/w). Inclusion of SFP lead to a significant reduction in starch digestibility and hence the potential glycaemic values of pasta (experimental pasta being up to 143% lower than control values). SFP addition to pasta increased the release of phenolic compounds from pasta during both a gastric digestion (179%) and pancreatic digestion ( 133%) in comparison to the control sample. At the same time, the antioxidant activity of the digested pasta was increased by up to 263% (gastric) and 190% (pancreatic) in comparison to durum wheat pasta alone. Interestingly, although protein levels increased with incorporation of SFP, the digestibility values of the protein decreased from 86.41% for the control pasta to 81.95% for 20% SFP pasta. This may indicate that there are interactions between phenolic and protein in the pasta samples which affect overall protein digestibility levels.

Lipophilic oligonucleotide derivatives are a potent approach to the intracellular delivery of nucleic acids. The binding of these derivatives to serum albumin is a major determinant of their fate in the body, as its structure contains several sites of high affinity for hydrophobic compounds. This study focuses on the features of self-association and non-covalent interactions with human serum albumin of novel self-penetrating oligonucleotide derivatives. The study revealed that the introduction of a triazinyl phosphoramidate modification bearing two dodecyl groups at the 3' end region of the oligonucleotide sequence has a negligible effect on its affinity for the complementary sequence. Dynamic light scattering verified that the amphiphilic oligonucleotides under study can self-assemble into micelle-like particles ranging from 8 to 15 nm in size. The oligonucleotides with dodecyl groups form stable complexes with human serum albumin with a dissociation constant of approximately 10-6 M. The oligonucleotide micelles are simultaneously destroyed upon binding to albumin. Using an electrophoretic mobility shift assay and affinity modification, we examined the ability of DNA duplexes containing triazinyl phosphoramidate oligonucleotides to interact with Ku antigen and PARP1, as well as the mutual influence of PARP1 and albumin or Ku antigen and albumin upon interaction with DNA duplexes. These findings, together with the capability of dodecyl-containing derivatives to effectively penetrate different cells, such as HEK293 and T98G, indicate that the oligonucleotides under study can be considered as a platform for the development of therapeutic preparations with a target effect.

mRNA vaccines have attracted widespread research attention with clear advantages in terms of molecular flexibility, rapid development, and potential for personalization. However, current mRNA vaccine platforms have not been optimized for induction of CD4/CD8 T cell responses. In addition, mucosal administration of mRNA based on lipid nanoparticle technology faces challenges in clinical translation. In contrast, adenovirus-based vaccines induce strong T-cell responses and have been approved for intranasal delivery. To leverage the inherent strengths of both the mRNA and adenovirus platforms, we developed a novel modular adenoviral mRNA delivery platform based on Tag/Catcher bioconjugation. Specifically, we engineered adenoviral vectors integrating Tag/Catcher proteins at specific locales on the Ad capsid proteins, allowing us to anchor mRNA to the surface of engineered Ad viruses. In proof-of-concept studies, the Ad-mRNA platform successfully mediated mRNA delivery and could be optimized via the highly flexible modular design of both the Ad-mRNA and protein bioconjugation systems.

Changes in epitranscriptome with N6‐methyladenine (m6A) modification could be involved in the development of multiple diseases, which might be a prevalent modification of messenger RNAs (mRNAs) in eukaryotes. The m6A modification might be performed through the action of methyltransferases, demethylases, and methylation-binding proteins. Importantly, the m6A methylation may be associated with various neurological disorders including Alzheimer's disease (AD), Parkinson's disease (PD), depression, aging related diseases, and/or aging itself. In addition, the m6A methylation might functionally regulate the eukaryotic transcriptome by influencing splicing, export, subcellular localization, translation, stability, and decay of mRNAs. Neurodegenerative diseases may possess a wide variety of phenotypes depending on the neurons that degenerate at occasions. Interestingly, increasing number of evidences have indicated that m6A modification could modulate the expression of autophagy-related genes and promote autophagy in neuronal cells. Oxidative stresses such as reactive oxygen species (ROS) could stimulate the m6A RNA methylation, which may be also related to the regulation of autophagy and/or the development of neurodegenerative diseases. Both m6A modification and autophagy could also play critical roles in regulating health condition of neurons. Therefore, a comprehensive understanding of the m6A and autophagy relationship in human diseases may benefit in developing therapeutic strategies in the future. This paper reviews advances in the understanding of the regulatory mechanisms of m6A modification in the occurrence and development of neurodegenerative diseases and/or aging, discussing the possible therapeutic procedures related to mechanisms of m6A RNA methylation and autophagy.

Fragile X syndrome (FXS) is a neurodevelopmental disorder associated with a wide range of cognitive, behavioral and medical problems. It arises from the silencing of the fragile X mental retardation 1 (FMR1) gene, and consequently, in the absence of its encoded protein, FMRP (Fragile X Mental Retardation Protein). FMRP is a ubiquitously expressed and multifunctional RNA-binding protein, primarily considered as a translational regulator. Pre-clinical studies of the past two decades have therefore focus on this function to relate FMRP’s absence to the molecular mechanisms underlying FXS physiopathology. Based on these data, successful pharmacological strategies were developed to rescue fragile X phenotype in animal models. Unfortunately, these results did not translate into human, as clinical trials using same therapeutic approaches did not reach the expected outcomes. These failures highlight the need to put into perspectives the different functions of FMRP in order to get a more comprehensive understanding of FXS pathophysiology. In this review, FMRP’s involvement on noteworthy molecular mechanisms are pointed out; ultimately contributing to various biochemicals alterations composing the fragile X phenotype.

DDX3 is an ATP-dependent RNA helicase involved in multiple cellular activities, including RNA metabolism and innate immunity. DDX3 is known to assist the replication of some viruses while restricting some others through direct interaction with the viral proteins. However, the role of DDX3 in the replication of the hepatitis E virus (HEV) is unknown. In this study, DDX3 is shown to interact with the HEV capsid protein and provide an indispensable role in HEV replication. The DDX3 C-terminal domain was demonstrated to interact with the capsid protein, which was previously demonstrated to inhibit the production of type I interferons. Knockdown of DDX3 compromised the capsid protein-mediated blockage of interferon induction. Notably, DDX3 silencing led to a significant reduction in HEV replication. Also, the ATPase activity of DDX3 is required for the HEV replication as an ATPase-null mutant DDX3 failed to rescue the viral replication in the DDX3-silenced cells. These results demonstrate a pro-viral role of DDX3 in HEV replication, providing further insights into the virus-cell interactions.

This study presents a methodology to reveal traces of viral particles, as aerosol with known chemical and molecular structure, in a sample by means of photon and electron interactions. The method is based on Monte Carlo simulations and on the analysis of photon-electron fluxes-spectra through energy channels counts as a function of different aerosol viral concentrations in the air sample and looking at the peculiar photon/electron interactions with the potential abnormal atomic hydrogen (H), oxygen (O), carbon (C), and phosphorus (P) compositions present in the air sample as a function of living and nonliving matter with PO4 group RNA/DNA strands in a cluster configuration.

Ribosome assembly is one of the most fundamental processes in gene expression and has served as a playground to investigate the molecular mechanisms of how protein-RNA complexes (RNPs) assemble. The bacterial ribosome is composed of around 50 ribosomal proteins several of which are co-transcriptionally assembled on a ~4,500 nucleotides long pre-rRNA transcript that is further processed and modified during transcription, the entire process taking around 2 minutes in vivo and assisted by dozens of assembly factors. How this complex molecular process works so efficiently to produce an active ribosome has been investigated over decades and has resulted in the development of a plethora of novel approaches that can also be used to study the assembly of other RNPs. Here we review biochemical, structural and biophysical methods that have been developed and integrated to provide a detailed and quantitative understanding of this complex and intricate molecular process of assembly. We also discuss emerging cutting-edge approaches that could be used in the future to study how transcription, rRNA processing, cellular factors and the native cellular environment shape ribosome assembly and RNP assembly at large.

Myelodysplastic syndromes or neoplasms (MDS) are a heterogeneous group of myeloid clonal disorders characterized by peripheral blood cytopenias, blood and marrow cell dysplasia and an increased risk for evolution to acute myeloid leukemia (AML). Non-coding RNAs, especially microRNAs and long non-coding RNAs serve as regulators of normal and malignant hematopoiesis and have been implicated in carcinogenesis. This review will present a comprehensive summary of the biology and role of non-coding RNAs, including the less studied circRNA, siRNA, piRNA, and snoRNA as potential prognostic and/or predictive biomarkers or therapeutic targets in MDS.

All life on Earth uses three integrated molecular systems in which genetic information contained in DNA base sequences is transmitted to ribosomes by RNA and a genetic code, then translated into the amino acid sequences of structural and catalytic proteins. Therefore, the most important point for understanding the origin of life is to determine how such systems could emerge from random processes on the early Earth. In this review, two alternatives are compared: the RNA world hypothesis and the [GADV]-protein world hypothesis. [GADV] refers to four amino acids, Gly [G], Ala [A], Asp [D] and Val [V] that are conserved in the amino acid sequences of many common proteins. Here I will argue that the origins of the three primary processes required for life to begin can be better explained by the GADV hypothesis than the RNA world hypothesis. The GADV hypothesis also incorporates a conversion process by which random polymers can evolve into proteins with ordered sequences.

Nuclear export of viral mRNAs, is an essential step in the HIV replication cycle. This role is played by a small regulatory protein of HIV-1 called Rev.The N-terminal region of Rev contains an arginine-rich sequence. The arginine-rich motif (ARM) is located between amino acids 38-50 and forms an alpha-helical secondary structure. Expression of the structural proteins of human immunodeficiency virus type 1 requires the direct interaction of multiple copies of the viral Rev protein with its highly structured RNA target sequence, the Rev Response element (RRE). The major viral proteins are not produced if this transport of RNA is stopped. Therefore, knowledge of Rev structure is essential for understanding of its cooperative binding to the RRE, for understanding the mechanism of HIV infection and for the development of antiviral drugs that interfere with Rev’s essential functions and for acknowledgment of good candidate drugs for treatment of AIDS. To understand how REV interact with RRE element of HIV-RNA and its formation of oligomeric complex it is better to characterize the domain wise structure of REV with regard in function of each domain. Due to lack of structural data on Rev no single compound is reported as inhibitor of REV expect antiviral drugs. Identification of a high-affinity RNA-binding site for the human immunodeficiency virus type 1 Rev Protein is much more important. The ARM is a highly specific sequence which allows for the multimerization of Rev and also binding of REV with RNA. Here we are first time exploring the structural characteristics of REV protein both in free form and in complex with RNA at domain function level especially explore the role of ARM motif in REV HIV-1 protein as RNA binding sites by molecular dynamics (MD) simulation and homology modeling studies. Results indicate that the arginine-rich motif (ARM) is crucial in stability of this complex. The residues ARG38, 39, 41, 43, 44, 48, 50, and ASN40 are most interacting with nucleobases of RRE in Crystal structure of Rev and Rev-response-element RNA complex. Our study plays a major role in elaboration of binding of RNA with REV and pave the way for further investigation for therapeutically agent for HIV.

The origin of life has not been solved as yet, in spit of the time passage more than thirty years from publication of RNA world hypothesis by W. Gilbert (1986), which is based on the “gene/replicator--first” theory. On the contrary, I have proposed [GADV]-protein world hypothesis (GADV hypothesis), assuming that life emerged from [GADV]-protein world, which is grounded on the “protein/metabolism-first” theory. However, two weak points of protein world hypothesis, (i) protein cannot be produced without gene, and (ii) protein cannot be self-replicated, have been frequently pointed out by supporters of RNA world hypothesis. Then, I examined whether the two weak points could be overcome by GADV hypothesis or not. From the results, it was confirmed that (i) [GADV]-protein could be pseudo-replicated in the absence of gene owing to protein 0^th-order structure or [GADV]-amino acids, and (ii) the replication ability is not always required from the beginning but it is sufficient to acquire it at some time point until the emergence of life. Thus, it was concluded that life emerged as [GADV]-protein world hypothesis, which is grounded on the “protein/metabolism-first” theory, expects.

The effects of hypothermia on neonatal encephalopathy may vary regionally, spatially, and cytopathologically in the gyrencephalic neocortex with manifestations potentially influenced by seizures that alter the severity and distribution of neuropathology. We developed a neonatal piglet survival model of hypoxic-ischemic (HI) encephalopathy and hypothermia with continuous encephalographic (cEEG) monitoring for seizures to study injury in neocortex. Neonatal piglets were randomized to naïve, HI-normothermia (NT), overnight hypothermia (HT) initiated 2 hours after HI, sham-NT, or sham-HT treatments. Some piglets within sham and HI groups received cEEG monitoring during recovery. Survival was 2-7 days (piglets were unmedicated and those with poor recovery and unresolving seizures were euthanized early); there was no differences in survival among groups (p&gt;0.078). Neuropathology was assessed by hematoxylin and eosin staining and immunohistochemistry for RNA Binding FOX-1 Homolog 3 (Rbfox3/NeuN). Normal and ischemic-necrotic neurons were counted (layers II-VI collectively) in somatosensory, motor, and prefrontal cortices, identified by cytoarchitecture and connectomics, and in inferior parietal cortex by layer. Seizure burden was determined. HI-NT piglets had reduced normal/total neuron ratio and increased ischemic-necrotic/total neuron ratio relative to naïve, sham-NT, and sham-HT piglets in anterior and posterior motor and somatosensory cortices. Frontal cortex was vulnerable in the prefrontal lateral bank after HI-NT with ischemic-necrosis. HI-HT piglets had higher normal/total neuron ratios and lower ischemic-necrotic/total neuron ratios than HI-NT piglets in anterior/posterior motor and somatosensory cortices. Total normal neuron density in layer III of inferior parietal cortex was reduced in HI-NT piglets compared to sham piglets and was protected by HT. Laminar analysis of Rbfox3 in somatosensory cortex revealed three types of neurons: Rbfox3-positive/normal, Rbfox3-positive/ischemic-necrotic, and Rbfox3-depleted. HI piglets had increased Rbfox3-depleted/total neuron ratio in layers II and III compared to sham-NT. Cortical neuron Rbfox3-depletion was partly rescued by HT. Seizure burden was more severe in HI piglets compared to sham piglets, but HI-NT and HI-HT piglets were similar. We conclude that 1) the neonatal piglet neocortex has a suprasylvian spatial vulnerability to HI and seizures; 2) HT protects against neuropathology in functionally different regions of the neonatal gyrencephalic neocortex; 3) neurons in neonatal neocortex have a limited cytopathology repertoire seen by H&amp;E staining; 4) higher seizure burden correlates with more ischemic-necrotic neurons in somatosensory cortex; 5) seizure presence associates with damage spread to inferior parietal cortex; 6) seizure presence is insensitive to HT, and 7) Rbfox3 immunophenotyping identifies a novel neuronal RNA splicing protein nuclear-depletion pathology that appears reversible by HT. This work demonstrates that HT protection of neocortex in neonatal HI is topographic and laminar, seizure unmitigating, and restores depleted neuronal RNA splicing factor.

Translation of the genetic code into proteins is the main process across all life and ribosomes are ancient cellular machines uniquely enabling this information transformation. We provide a brief overview of the recent advances in linking the ribosomal structure and evolution. Based on these insights into ribosomal organisation across time, we propose that early replication and protein biosynthesis functions were inseparable and in fact were performed by the same ancient RNA molecule, the riboreplisome. Riboreplisome hypothesis helps to address issues of non-Darwinian evolution and complicated starting point that are characteristic to the RNA world, protein world and RNA:protein mixed co-development theories. We suggest that the riboreplisome is the missing link and a molecular machine connecting chemical and biological evolution paths, by being capable of basic genetic and feature selection functions in a cell- or cell-free setting. The riboreplisome hypothesis allows ease of sequential, genetically uninterrupted emergence and sophistication of the genetic code and its decoding machinery, and provides plausible explanations to the origins of the three main RNA types involved in the decoding: the ribosomal, transfer and messenger RNA. Furthermore, riboreplisome can help explaining the co-evolution of aminoacylation machinery, the driving force behind selective gene transcription and expression, and the cell-like compartmentalisation. While we may never find the original riboreplisome again, we might continue to discover different molecular remnants of its prior existence across the existing biological RNA, which, once identified or resurrected, can be useful in synthetic biology applications.

Current knowledge of the RNA world indicates two different genetic codes being present throughout the living world. In contrast to non-coding RNAs that are built of repetitive nucleotide syntax, the sequences that serve as templates for proteins share – as main characteristics – a non-repetitive syntax. The differences in their syntax structure is coherent with the difference of the functions they represent. Whereas non-coding RNAs build groups that serve as regulatory tools in nearly all genetic processes, the coding sections represent the evolutionarily successful function of the genetic information storage medium. The DNA genomes themselves are rather inactive, whereas the non-coding RNA domain is highly active, even as non-random genetic innovation operators. This indicates that repetitive syntax is the essential pre-requisite for RNA interactions to install variable RNA-group-identities, whereas the non-repetitive syntax serves as a stable conservation tool for successful selection processes out of RNA-groups cooperation and competition. The interaction opportunities of RNA loops with repetitive syntax are higher than with non-repetitive ones. Interestingly, these two genetic codes resemble the function of all natural languages, i.e., (a) everyday language use for organization and coordination of biotic group behavior, and (b) artificial (instrumental) language use for conservation of blueprints for complex protein-body constructions.

The transfer of protein-encoding genetic information from DNA to RNA to protein, a process formalized as the “Central Dogma of Molecular Biology”, has undergone a significant evolution since its inception. It was amended to account for the information flow from RNA to DNA, the reverse transcription, and for the information transfer from RNA to RNA, the RNA-dependent RNA synthesis. These processes, both potentially leading to protein production, were initially described only in viral systems, and although RNA-dependent RNA polymerase activity was shown to be present, and RNA-dependent RNA synthesisfound to occur, in mammalian cells, its function was presumed to be restricted to regulatory. However, recent results, obtained with multiple mRNA species in several mammalian systems, strongly indicate the occurrence of protein-encoding RNA to RNA information transfer in mammalian cells. It can result in the rapid production of the extraordinary quantities of specific proteins as was seen in cases of terminal cellular differentiation and during cellular deposition of extracellular matrix molecules. A malfunction of this process may be involved in pathologies associated either with the deficiency of a protein normally produced by this mechanism or with the abnormal abundanceof a protein or of its C-terminal fragment. It seems to be responsible for some types of familial thalassemia and may underlie the overproduction of beta amyloid in sporadic Alzheimer’s disease. The aim of the present article is to systematize the current knowledge and understanding of this pathway. The outlined framework introduces unexpected features of the mRNA amplification such as its ability to generate polypeptides non-contiguously encoded in the genome, its second Tier, a physiologically occurring intracellular polymerase chain reaction, iPCR, a Two-Tier Paradox and RNA Dark Matter. RNA-dependent mRNA amplification represents a new mode of genomic protein-encoding information transfer in mammalian cells. Its potential physiological impact is substantial, it appears relevant to multiple pathologies and its understanding opens new venues of therapeutic interference, it suggests powerful novel bioengineering approaches and its further rigorous investigations are highly warranted.

The current SARS- CoV-2 pandemic underscores the importance of understanding the evolution of RNA genomes. While RNA is subject to the formation of similar lesions as DNA, the evolutionary and physiological impacts RNA lesions have on viral genomes are yet to be characterized. Lesions that may drive the evolution of RNA genomes can induce breaks that are repaired by recombination or can cause base substitution mutagenesis, also known as base editing. Over the past decade or so, base editing mutagenesis of DNA genomes has been subject to many studies, revealing that exposure of ssDNA is subject to hypermutation that is involved in the etiology of cancer. However, base editing of RNA genomes has not been studied to the same extent. Recently hypermutation of single-stranded RNA viral genomes have also been documented though its role in evolution and population dynamics. Here, we will summarize the current knowledge of key mechanisms and causes of RNA genome instability covering areas from the RNA world theory to the SARS- CoV-2 pandemic of today. We will also highlight the key questions that remain as it pertains to RNA genome instability, mutations accumulation, and experimental strategies for addressing these questions.

At the molecular level, aging is often accompanied by dysfunction of stress-induced membrane-less organelles (MLOs) and changes in their material state. In this work, we analyzed the proteins included in the proteome of stress granules (SGs) and P-bodies for their tendency to transform the material state of these MLOs. Particular attention was paid to proteins whose gene expression changes during replicative aging. It was shown that the proteome of the studied MLOs practically does not differ in the analyzed characteristics and consists of completely or partially intrinsically disordered proteins, 30 - 40% of which are potentially capable of liquid-liquid phase separation (LLPS). At the same time, the proportion of proteins capable of spontaneous LLPS is relatively small, which indicates the leading role of nucleic acids in the biogenesis of these membrane-less organelles. Proteins whose gene expression changes during the transition of human cells to a senescent state make up about 20% of the studied proteomes. There is a statistically significant increase in the number of positively charged proteins in both datasets studied compared to the complete proteomes of these organelles. An increase in the relative content of DNA-, but not RNA-binding proteins, was also found in the stress-granules dataset with senescence-related processes. Among SG proteins potentially involved in senescent processes, there is an increase in the abundance of potentially amyloidogenic proteins compared to the whole proteome. The hnRPDL protein has the highest degree of disorder and highest propensity for LLPS among such proteins, which allows us to consider it as “potentially dangerous.” Proteins common to SGs and P bodies, potentially involved in processes associated with senescence, form clusters of interacting proteins. The largest cluster is represented by RNA-binding proteins involved in RNA processing and translation regulation. These data indicate that SG proteins, but not proteins of P-bodies, are more likely to transform the material state of MLOs. Furthermore, these MLOs can participate in processes associated with aging in a coordinated manner.

Vaccine development against SARS-CoV-2 has been highly successful in slowing down the COVID-19 pandemic. A wide spectrum of approaches including vaccines based on whole viruses, protein subunits and peptides, viral vectors, and nucleic acids have been developed in parallel. For all types of COVID-19 vaccines, good safety and efficacy have been obtained in both preclinical animal studies and in clinical trials in humans. Moreover, emergency use authorization has been granted for all types of COVID-19 vaccines. Although high safety has been demonstrated, rare cases of severe adverse events have been detected after global mass vaccinations. Emerging SARS-CoV-2 variants possessing enhanced infectivity have affected vaccine protection efficacy requiring re-design and re-engineering of novel COVID-19 vaccine candidates. Furthermore, insight into preparedness against emerging SARS-CoV-2 variants and other future viral infections is given.

In this study, an analysis was conducted on 16 durum wheat genotypes cultivated under seven different crop management systems. The purpose was to investigate the influence of Genotype (G), Environment (E), and Genotype by Environment interactions (GxE) on the content of total free phenolic compounds (TPC) and the antioxidant capacity (AC) of durum wheat in Mediterranean environments and identify their adaptability in climatic change. Additionally, possible correlations between TPC and AC with protein content (PC), and vitreous-kernel percentage (VKP) of the samples were examined. The results indicated significant impacts from E, G, and GxE on both TPC and AC. Among the E, the mean values for TPC, ABTS, DPPH, and FRAP values were recorded at 48.8 mg TE/100 g dw, 121.3 mg TE/100 g dw, 23.0 mg TE/100 g dw, and 88.4 mg TE/100 g dw, respectively. Environments subjected to splitting topdressing N fertilization consistently showed low values, while the late sowing ones possessed high values. The Organic environment maintained a stable position in the middle across all measurements. The predominant influence was attributed to GxE, as indicated by the order GxE &gt;E&gt;G for ABTS, DPPH, and FRAP while for TPC was E&gt;GxE&gt;G. A comprehensive exploration of GxE interaction holds the potential to improve the total free phenolics content and antioxidant capacity of durum wheat within wheat farming systems. Achieving this enhancement involves the meticulous selection of cultivars that not only display high values but also exhibit stability across diverse conditions. Noteworthy genotypes for TPC include G5, G7 and G10, for ABTS include G3, G5 and G7, and for protein include G1, G9, and G16. G7 and G5 had high presence of frequency with G7 being the closest genotype to the ideal for both TPC and ABTS. These results suggest that the cultivation period as well as the nitrogen fertilization and its application method have a significant impact on the various antioxidant properties of durum wheat. This study holds significant importance as it represents the first comprehensive exploration of the impact of various E on the TPC and AC in durum wheat, with a special emphasis on the crop management. Notably, the significance of several statistical tools is also highlighted, such as the GGE biplot, Pi, and KR, in identifying superior genotypes, with the GGE biplot being highly valuable since it offers a visual representation of the study's outcomes.

Expression analysis of small noncoding RNA (sRNA), including microRNA, piwi-interacting RNA, small rRNA-derived RNA, and tRNA-derived small RNA, is a novel and quickly developing field. Despite a range of proposed approaches, selecting and adapting a particular pipeline for transcriptomic analysis of sRNA remains a challenge. This paper focuses on the identification of the optimal pipeline configurations for each step of human sRNA analysis, including reads trimming, filtering, mapping, transcript abundance quantification and differential expression analysis. Based on our study, we suggest the following parameters for analysis of human sRNA in relation to categorical analyses with two groups of biosamples: (1) trimming with the lower length bound = 15 and the upper length bound = \(Read\ length - 40\% Adapter\ length\); (2) mapping on a reference genome with bowtie aligner with one mismatch allowed (-v 1 parameter); (3) filtering by mean threshold > 5; and (4) analyzing differential expression with DESeq2 with adjusted p-value < 0.05 or limma with p-value < 0.05 if there is very little signal and few transcripts.

RNA silencing serves key roles in a multitude of cellular processes, including development, stress responses, metabolism, and maintenance of genome integrity. Dicer, Argonaute (AGO), double-stranded RNA binding (DRB), RNA-dependent RNA polymerase (RDR) and DNA-dependent RNA polymerases known as Pol IV and Pol V form core components to trigger RNA silencing. Common bean (Phaseolus vulgaris) is an important staple crop worldwide. In this study, we aimed to unravel the components of the RNA-guided silencing pathway in this non-model plant taking advantage of the availability of two genome assemblies of Andean and Meso-American origin. We identified six PvDCLs, thirteen PvAGOs, 10 PvDRB, 5 PvRDR, in both genotypes, suggesting no recent gene amplification or deletion after the gene pool separation. In addition, we identified one PvNRPD1 and one PvNRPE1 encoding the largest subunits of Pol IV and Pol V, respectively. These genes were categorized into subgroups based on phylogenetic analyses. Comprehensive analyses of gene structure, genomic localization and similarity among these genes were performed. Their expression patterns were investigated by means of expression models in different organs using online data and quantitative RT-PCR after pathogen infection. Several of the candidate genes were up-regulated after infection with the fungus Colletotrichum lindemuthianum.

After a long limbo, RNA has gained its credibility as a druggable target, fully earning its de-served role in the next-generation area of pharmaceutical R&D. We have recently probed the Trans-Activation Response element (TAR), a RNA stem–bulge–loop domain of the HIV-1 genome with bis-3-chloropiperidines (B-CePs), and revealed the compounds unique behavior in stabiliz-ing TAR structure, thus impairing in vitro the chaperone activity of the HIV-1 nucleocapsid (NC) protein. Seeking to elucidate the determinants of B-CePs inhibition, we have further characterized here their effects on the target TAR and its NC recognition, while developing quantitative analyti-cal approaches for the study of multicomponent RNA-based interactions.

This review will explore the four major pillars required for design and development of an saRNA vaccine: antigen design, vector design, non-viral delivery systems, and manufacturing (both saRNA and lipid nanoparticles (LNP)). In will report on the major innovations, preclinical and clinical data reported in the last five years and will discuss future prospects.

Long non-coding RNAs (lncRNAs) have been found to be involved in many biological processes, including the regulation of cell differentiation, but a complete characterization of lncRNA is still lacking. Additionally, there is evidence that lncRNAs interact with ribosomes, raising questions about their functions in cells. Here, we used a developmentally staged protocol to induce cardiogenic commitment of hESCs and then investigated the differential association of lncRNAs with polysomes. Our results identified lncRNAs in both the ribosome-free and polysome-bound fractions during cardiogenesis and showed a very well-defined temporal lncRNA association with polysomes. Clustering of lncRNAs was performed according to the gene expression patterns during the five timepoints analyzed. In addition, differential lncRNA recruitment to polysomes was observed when comparing the differentially expressed lncRNAs in the ribosome-free and polysome-bound fractions or when calculating the polysome-bound vs ribosome-free ratio. The association of lncRNAs with polysomes could represent an additional cytoplasmic role of lncRNAs, e.g., in translational regulation of mRNA expression.

Noncoding RNA regions have emerged as pivotal players in the regulation of various cellular processes, and their involvement in HIV/AIDS pathogenesis is an evolving field of research. This review article delves into the intricate interplay between noncoding RNA regions and AIDS, shedding light on the diverse mechanisms by which these RNA molecules influence viral replication, immune responses, and disease progression. The review highlights recent discoveries, potential therapeutic applications, and the challenges ahead, illustrating the promising prospects of noncoding RNA research in the context of HIV/AIDS.

Membrane-coupled RNA transport is an emerging theme in fungal biology. This review focuses on the RNA cargo and mechanistic details of transport via two inter-related sets of organelles: endosomes and extracellular vesicles for intra- and intercellular RNA transfer. Simultaneous transport and translation of messenger RNAs (mRNAs) on the surface of shuttling endosomes is a conserved process pertinent to highly polarised eukaryotic cells, such as hyphae or neurons. Here we detail the endosomal mRNA transport machinery components and mRNA targets of the core RNA-binding protein Rrm4. Extracellular vesicles (EVs) are newly garnering interest as mediators of intercellular communication, especially between pathogenic fungi and their hosts. Landmark studies in plant-fungus interactions indicate EVs as a means of delivering various cargos, most notably small RNAs (sRNAs), for cross-kingdom RNA interference. Recent advances and implications of the nascent field of fungal EVs are discussed and potential links between endosomal and EV-mediated RNA transport are proposed.

RNA interference (RNAi) is one of the main mechanisms for disease resistance and small RNA production in plants. The main proteins involved in RNAi include Dicer-like (DCL), RNA-dependent RNA polymerase (RDR), double-stranded RNA-binding (DRB), and Argonaute (AGO). Juglandaceae contains a variety of important woody plants, and walnuts are one of the four major woody plant groups and one of the four major dried fruits in the world. To clarify the evolution and functional differentiation of RNAi-related proteins in the walnut (Juglans regia) genome, this study integrated various web resources from gene family acquisition to structural analysis and transcriptome data to correlate walnuts and their congeners. The walnut genome has 5 DCL, 13 RDR, 15 DRB and 15 AGO genes, similar genes encoding conserved protein structural domains and conserved motifs with similar subcellular localization. Walnut AGO proteins are classified into three classes and seven subclasses. The DCL is divided into four categories, while RDR is mainly divided into four categories, and DRB can be divided into six categories. The exception is that the copy number of walnut RDR1 is 9, in which seven RDR1 are distributed in clusters on chromosome 16. Purifying selection drove the formation of walnut genes, but protein classes were subjected to varying degrees of purifying selection. Additionally, these results showed some similarity in other plants of the walnut family. Moreover, different RNAi-related genes of walnut produced abundant selective expression in response to different tissues and stresses. In this study, DCL, RDR, DRB and AGO gene families were identified and analysed in the genome of the walnut family for the first time and preliminarily examined the evolution, structure and expression characteristics of these families to provide a preliminary basis for the evolution of the walnut RNAi pathway and breeding research.

RNA extraction from human cumulus cells (CCs) is considered challenging due to low cell’s pop-ulation and small cell. The successful gene expression analysis from cumulus cells ultimately de-pends on an excellent concentration of Ribonucleic acid (RNA) yield by an appropriate technique. Our study aims to consolidate appropriate cumulus cell collection method, preparation and ex-traction techniques to ensure a good yield of RNA for better gene expression analysis. From the results obtained, the optimal strategy found involved mechanical denudation without an enzy-matic process, followed by the use of RNA stabilizer prior to RNA extraction. Subsequently, uti-lizing a smaller homogenizer, preferably manual pressure control with filter (Bio-Masher III®) is paramount for a pure and homogenize sample. Subsequently, during RNA extraction, the use of RNeasy micro kit and RNA carriers is recommended. These sample collection, preparation, and extraction techniques yielded an excellent-quality RNA concentration and successful gene expres-sion analysis. We also conducted a systematic review to consolidate our findings with current evidence of RNA extraction technique. Our results added a value as a current strategy for opti-mizing RNA extraction in CCs for experimental studies. Thus, our findings can contribute to for-mulating a better strategy for optimizing the RNA extraction in CCs for transcriptomic studies.

A definitive transcriptome atlas for the non-coding expressed elements of pathogenic mycobacteria does not exist. Incomplete lists of non-coding transcripts can be obtained for some of the reference genomes (e.g. Mycobacterium tuberculosis H37Rv) but to what extent these transcripts have homologues in closely related species or even strains is not clear. This has implications for the analysis of transcriptomic data; non-coding parts of the transcriptome are often ignored in the absence of formal, reliable annotation. Here, we review the state of our knowledge of non-coding RNAs in pathogenic mycobacteria, emphasising the disparities in the information included in commonly used databases. We then proceed to review ways of combining computational solutions for predicting the non- coding transcriptome with experiments that can help refine and confirm these predictions.

Plant pathogenic fungi are the largest group of disease-causing agents on crop plants and represent a persistent and significant threat to agriculture worldwide. Conventional approaches based on the use of pesticides raise social concern for the impact on the environment and human health and alternative control methods are urgently needed. The rapid improvement and extensive implementation of RNAi technology for various model and non-model organisms has provided the initial framework to adapt this post-transcriptional gene silencing technology for the management of fungal pathogens. In this review, we describe exogenous RNAi involved in plant pathogenic fungi and discuss small RNA production, formulation, and RNAi delivery methods. We explore some challenges with possible solutions. Furthermore, exogenous RNAi holds great potential for RNAi-mediated plant pathogenic fungal disease control.

RNA interference is a highly conserved process in which non-coding small RNAs (sRNA) modulate gene expression at the post-transcriptional level influencing plant growth and development. Dicer-like (DCL), Argonaute (AGO) and RNA-dependent RNA polymerase (RDR) are the core elements involved in gene silencing and their gene families have been explored in many plants. However, these genes and their response to abiotic stresses have not yet been well in wheat. In this study, 82 AGO, 31 DCL, and 31 RDR genes were identified and phylogenetic analysis of these proteins showed that clustered into ten, four and four clades respectively. RNA-seq analysis revealed constitutive expression of AGO1, AGO9, and DCL2 family expression analysis in tissues under normal and stress conditions, whereas RDR1 which is known to engage in siRNA biogenesis showed higher expression levels in wheat leaf tissues. Our findings build the foundation for comparative genomics analyses of RNA silencing elements in cereal crops, as well as new insights into the functional complexity of RNA silencing in wheat stress responses, which is critical for understanding the processes underlying wheat stress responses.

Lung cancer is a common malignant tumor of the lung and the leading cause of cancer mortality worldwide. Non-small-cell lung cancer (NSCLC) accounts for 80%–85% of lung cancer, 40% of NSCLCs will have spread beyond the lungs by the time it is diagnosed. Long non-coding RNA (LncRNA) prostate androgen-regulated transcript 1 (PART-1) was reported that promote the development of several cancers. In the current study, we conducted experiments to investigate the role of PART-1 in the proliferation, invasion, and migration of NSCLC. The expression level of the PART-1 gene increased significantly in the NSCLC cell lines, including A549, H1229, H1650, H1975, and PC9. Knocking down of PART-1 inhibited the proliferation, invasion, and migration of A549 cells, moreover, decreased the tumor proliferation in nude mice. We confirmed that PART-1 targeted miR-204-3p directly, and miR-204-3p targeted the insulin-like growth factor binding protein 2 (IGFBP-2) directly. Furthermore, we discovered that PART-1 involved the NSCLC progression by regulating the miR-204-3p-targeted IGFBP-2 pathway. LncRNA PART-1 might be a target for treating NSCLC, and a warning sign of diagnosis of early lung cancer.

The ribosome is a macromolecular complex composed of RNA and proteins that interact through an integrated and interconnected network to preserve its ancient core activities. In this review, we emphasize the pivotal role played by RNA-binding proteins as a driving force in the evolution of the current form of the ribosome, underscoring their importance in ensuring accurate protein synthesis. This category of proteins includes both ribosomal proteins and ribosome biogenesis factors. Impairment of their RNA-binding activity can also lead to ribosomopathies, a group of disorders characterized by defects in ribosome biogenesis that are detrimental to protein synthesis and cellular homeostasis. A comprehensive understanding of these intricate processes is essential for elucidating the mechanisms underlying the resulting diseases and advancing potential therapeutic interventions.

Non-coding RNAs (ncRNAs) comprise a diverse class of non-protein coding transcripts that regulate critical cellular processes associated with cancer. Advances in RNA-sequencing (RNA-Seq) have led to the characterization of non-coding RNA expression across different types of human cancers. Through comprehensive RNA-Seq profiling, a growing number of studies demonstrate that ncRNAs, including long non-coding RNA (lncRNAs) and microRNAs (miRNA), play central roles in progenitor B-cell Acute Lymphoblastic Leukemia (B-ALL) pathogenesis. Furthermore, due to their central roles in cellular homeostasis and their potential as biomarkers, the study of ncRNAs continues to provide new insight into the molecular mechanisms of B-ALL. This article reviews the ncRNA signatures reported for all B-ALL subtypes, focusing on technological developments in transcriptome profiling and recently discovered examples of ncRNAs with biologic and therapeutic relevance in B-ALL.

Prostate cancer (Pca) is a highly heterogeneous disease and the second more common tumor in males. Molecular and genetic profiles have been used to identify subtypes and guide therapeutic intervention. However, roughly 26% of primary Pca are driven by unknown molecular lesions. We use Principal Component Analysis (PCA) and custom RNAseq-data normalization to identify a gene expression signature which segregates primary PRAD from normal tissues. This Core-Expression Signature (PRAD-CES) includes 33 genes and accounts for 39% of data complexity along the PC1-cancer axis. The PRAD-CES is populated by protein-coding (AMACR, TP63, HPN) and RNA-genes (PCA3, ARLN1) sparsely found in previous studies, validated/predicted biomarkers (HOXC6, TDRD1, DLX1), and/or cancer drivers (PCA3, ARLN1, PCAT-14). Of note, the PRAD-CES also comprises six over-expressed LncRNAs without previous Pca association, four of them potentially modulating driver’s genes TMPRSS2, PRUNE2 and AMACR. Overall, our PCA capture 57% of data complexity within PC1-3. GO enrichment and correlation analysis involving major clinical features (i.e., Gleason Score, AR Score, TMPRSS2-ERG fusion and Tumor Cellularity) suggest that PC2 and PC3 gene signatures might describe more aggressive and inflammation-prone transitional forms of PRAD. Of note, surfaced genes may entail novel prognostic biomarkers and molecular alterations to intervene. Particularly, our work uncovered RNA genes with appealing implications on Pca biology and progression.

S100 proteins are multifunctional calcium-binding proteins of vertebrates that act intracellularly, extracellularly, or both, and are engaged in the progression of many socially significant diseases. Their extracellular action is typically mediated by the recognition of specific receptor proteins. Besides, recent studies indicate the ability of some S100 proteins to affect cytokine signaling through direct interaction with cytokines. S100P was shown to be the S100 protein most actively involved in interactions with some of four-helical cytokines. To assess selectivity of S100P protein binding to four-helical cytokines, we have probed interaction of Ca2+-bound recombinant human S100P with a panel of 32 four-helical human cytokines covering all structural families of this fold, using surface plasmon resonance spectroscopy. 22 cytokines from all families of four-helical cytokines are S100P binders with the equilibrium dissociation constants, Kd, ranging from 1 nM to 3 µM (below the Kd value for the S100P complex with the V domain of its conventional receptor, receptor for advanced glycation end products, RAGE). Molecular docking and mutagenesis studies revealed the presence in the S100P molecule of a cytokine-binding site, which overlaps with the RAGE-binding site. Since S100 binding to four-helical cytokines inhibits their signaling in some cases, the revealed ability of S100P protein to interact with ca 71% of the four-helical cytokines indicates that S100P may serve as a poorly selective inhibitor of their action.

Myelin protein zero (P0), a type I transmembrane protein, is the most abundant protein in peripheral nervous system (PNS) myelin – the lipid-rich, periodic structure that concentrically encloses long axonal segments. Schwann cells, the myelinating glia of the PNS, express P0 throughout their development until the formation of mature myelin. In the intramyelinic compartment, the immunoglobulin-like domain of P0 bridges apposing membranes together via homophilic adhesion, forming a dense, macroscopic ultrastructure known as the intraperiod line. The C-terminal tail of P0 adheres apposing membranes together in the narrow cytoplasmic compartment of compact myelin, much like myelin basic protein (MBP). In mouse models, the absence of P0, unlike that of MBP or P2, severely disturbs the formation of myelin. Therefore, P0 is the executive molecule of PNS myelin maturation. How and when is P0 trafficked and modified to enable myelin compaction, and how disease mutations that give rise to incurable peripheral neuropathies alter the function of P0, are currently open questions. The potential mechanisms of P0 function in myelination are discussed, providing a foundation for the understanding of mature myelin development and how it derails in peripheral neuropathies.

The SARS (severe acute respiratory syndrome)-CoV (Coronavirus)-2 S(spike)-protein mRNA/cDNA currently being used as vaccines are antigenic but not antigens against SARS-CoV-2, that causes COVID (Coronavirus Disease) -19. Furthermore, the mRNA and cDNA antigenic vaccines also have potentials for homologous as well as heterologous recombination, primarily into the somatic cell DNA of the vaccine recipients. On the contrary, a SARS-CoV-2 RBD-protein antigen, a part of the S-protein, will directly stimulate antibody production against SARS-CoV-2. Hence, a vaccine composed of SARS-CoV-2 RBD-protein as a safer, fast acting, and effective vaccine against SARS-CoV-2 and thus against COVID-19. This is also useful for some immune compromised individuals.

Transition between the unfolded and native states of the ordered globular proteins is accompanied by accumulation of several intermediates, such as pre-molten globule, wet molten globule, and dry molten globule. Structurally equivalent conformations can serve as native functional states of intrinsically disordered proteins. This overview captures the characteristics and importance of these molten globules in both structured and intrinsically disordered proteins. It also discusses examples of engineered molten globules. The formation of these intermediates under the conditions of macromolecular crowding and their interactions with nanomaterials are also reviewed.

Tobamoviruses are a group of plant viruses posing a significant threat to agricultural crops worldwide. In this review, we focus on plant immunity against tobamoviruses, including pattern-triggered immunity (PTI), effector-triggered immunity (ETI), RNA-targeting pathway, hormones and reactive oxygen species (ROS). Further, we highlight genetic resources for resistance against tobamoviruses in plant breeding and discuss future directions on plant protection against tobamoviruses.

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.

Long noncoding RNAs (lncRNAs) of virus origin accumulate in cells infected by many positive strand (+) RNA viruses to bolster viral infectivity. Their biogenesis mostly utilizes exoribonucleases of host cells that degrade viral genomic or subgenomic RNAs in the 5’-to-3’ direction until being stalled by well-defined RNA structures. Here we report a viral lncRNA that is produced by a novel replication-dependent mechanism. This lncRNA corresponds to the last 283 nucleotides of the turnip crinkle virus (TCV) genome, hence is designated tiny TCV subgenomic RNA (ttsgR). ttsgR accumulated to high levels in TCV-infected Nicotiana benthamiana cells when the TCV-encoded RNA-dependent RNA polymerase (RdRp), also known as p88, was overexpressed. Both (+) and (-) strand forms of ttsgR were produced in these cells in a manner dependent on the RdRp functionality. Strikingly, templates as short as ttsgR itself were sufficient to program ttsgR amplification, as long as the TCV-encoded replication proteins, p28 and p88, were provided in trans. Consistent with its replicational origin, ttsgR accumulation required a 5’ terminal G₃(A/U)₄ motif shown by others to be crucial for the replication of a TCV satellite RNA. More importantly, introducing a new G₃(A/U)₄ motif elsewhere in the TCV genome was alone sufficient to cause the emergence of another lncRNA. Collectively our results unveil a replication-dependent mechanism for the biogenesis of viral lncRNAs, thus suggesting that multiple mechanisms, individually or in combination, may be responsible for viral lncRNA production.

SARS-CoV-2 is the causative agent for the ongoing COVID19 pandemic, and this virus belongs to the Coronaviridae family. Like other members of this family, the virus possesses a positive-sense single-stranded RNA genome. The genome encodes for the nsp12 protein, which houses the RNA-dependent-RNA polymerase (RdRP) activity responsible for the replication of the viral genome. A homology model of nsp12 was prepared using the structure of the SARS nsp12 (6NUR) as a model. The model was used to carry out in silico screening to identify molecules among natural products, or FDA approved drugs that can potentially inhibit the activity of nsp12. This exercise showed that vitamin B12 (methylcobalamin) may bind to the active site of the nsp12 protein. A model of the nsp12 in complex with substrate RNA and incoming NTP showed that Vitamin B12 binding site overlaps with that of the incoming nucleotide. A comparison of the calculated energies of binding for RNA plus NTP and methylcobalamin suggested that the vitamin may bind to the active site of nsp12 with significant affinity. It is, therefore, possible that methylcobalamin binding may prevent association with RNA and NTP and thus inhibit the RdRP activity of nsp12. Overall, our computational studies suggest that methylcobalamin form of vitamin B12 may serve as an effective inhibitor of the nsp12 protein.

The telomerase RNA in yeasts is large, usually >1000 nt, and contains functional elements that have been extensively studied experimentally in several disparate species. Nevertheless, they are very difficult to detect by homology-based methods and so far have escaped annotation in the majority of the genomes of Saccharomycotina. This is a consequence of sequences that evolve rapidly at nucleotide level, are subject to large variations in size, and are highly plastic with respect to their secondary structures. Here we report on a survey that was aimed at closing this gap in RNA annotation. Despite considerable efforts and the combination of a variety of different methods, it was only partially successful. While 27 new telomerase RNAs were identified, we had to restrict our efforts to the subgroup Saccharomycetacea because even this narrow subgroup was diverse enough to require different search models for different phylogenetic subgroups. More distant branches of the Saccharomycotina still remain without annotated telomerase RNA.

In the past decade, RNA fragments derived from full length small nucleolar RNAs (snoRNAs) have been shown to be specifically excised and functional. These sno-derived RNAs (sdRNAs) have been implicated as gene regulators in a multitude of cancers, controlling a variety of genes post-transcriptionally via association with the RNA-induced silencing complex (RISC). In this review, we have summarized the literature connecting sdRNAs to cancer gene regulation. SdRNAs possess miRNA-like functions, and are able to fill the role of tumor-suppressor or tumor-promoter in a tissue context-dependent manner. Indeed, there are many miRNAs that are actually derived from snoRNA transcripts, meaning that they are truly sdRNAs and as such are included in this review. As sdRNAs are frequently discarded from ncRNA analyses, we emphasize that sdRNAs are functionally relevant gene regulators and likely represent an overlooked subclass of miRNAs. Based on the evidence provided by the papers reviewed here, we propose that sdRNAs deserve more extensive study to better understand their underlying biology and to identify previously overlooked biomarkers and therapeutic targets for a multitude of human cancers.

Reducing meat consumption is better for the environment. Unfortunately, commercial plant-based meat substitutes are often more expensive than meat and thus have not seen widespread adoption. This paper analyzes commercially-available spirulina, soy, pea, and brown rice protein isolates characteristics to provide data for non-meat protein cost reductions. Thermal and rheological properties, viscosity, density, and particle size distribution are analyzed for further study on alternative protein-based food processing. Differential scanning calorimetry analysis produced dry amorphous-shaped curves and paste curves with a more distinct endothermic peak. Extracted linear temperature ranges for processing in food production for spirulina was 70-90ºC; soy protein was 87-116ºC; pea protein was 67-77ºC; and brown rice protein was 87-97ºC. Viscosity analysis determined each protein material was shear-thinning and that viscosity increased with decreased water concentration, with rice being an exception to the latter trend. The obtained viscosity range for spirulina was 15,100-78,000cP; soy protein was 3,200-80,000cP; pea protein was 1,400-32,700cP; and brown rice protein was 600-3,500cP. The results indicate that extrusion is a viable method for further processing of the protein isolates as this technique has a large temperature operating range and variable screw speed. Data provided here can be used to make single or multi-component protein substitutes.

Myelin ensheathes selected axonal segments within the nervous system, resulting primarily in nerve impulse acceleration, as well as mechanical and trophic support for neurons. In the central and peripheral nervous systems, various proteins that contribute to the formation and stability of myelin are present, which also harbour pathophysiological roles in myelin disease. Many myelin proteins share common attributes, including small size, high hydrophobicity, multifunctionality, longevity, and intrinsic disorder. With recent advances in protein biophysical characterization and bioinformatics, it has become evident that intrinsically disordered proteins (IDPs) are abundant in myelin, and their flexible nature enables multifunctionality. Here, we review known myelin IDPs, their conservation, molecular characteristics and functions, and their disease relevance, along with open questions and speculations. We place emphasis on classifying the molecular details of IDPs in myelin and correlate these with their various functions, including susceptibility to post-translational modifications, function in protein-protein and protein-membrane interactions, as well as their role as extended entropic chains. We discuss how myelin pathology can relate to IDPs and which molecular factors are potentially involved.

Rabies is a neurological disease that causes between 40,000 and 70,000 deaths every year. Once a rabies patient has become symptomatic, there is no effective treatment for the illness, and in unvaccinated individuals, the case-fatality rate of rabies is close to 100%. French scientists Louis Pasteur and Émile Roux developed the first vaccine for rabies in 1885. If administered before the virus reaches the brain, the modern rabies vaccine imparts long-lasting immunity to the virus and saves more than 250,000 people every year. However, the rabies virus can suppress the host’s immune response once it has entered the cells of the brain, making death likely. This study aims to make use of disorder-based proteomics and bioinformatics to determine the impact that intrinsically disordered protein regions (IDPRs) in the proteome of the rabies virus have on the infectivity and lethality of the disease. This study uses the proteome of Rabies Lyssavirus (RABV) strain Pasteur Vaccins (PV), one of the best understood strains due to its use in the first rabies vaccine, as a model. The study suggests that the high levels of intrinsic disorder in the phosphoprotein (P-protein) and nucleoprotein (N-protein) allow them to participate in creation of the Negri bodies and help this virus suppress the antiviral immune response in the host cells. Additionally, the study suggests that there is a link between disorder in the matrix (M) protein and the modulation of viral transcription. The disordered regions in the M protein have a possible role in initiating viral budding within the cell. Furthermore, we checked the prevalence of functional disorder in a set of 37 host proteins directly involved in the interaction with the RABV proteins. The hope is that these new insights will aid in the development of treatments for rabies that are effective after infection.

Structural and biochemical studies have recently revealed a range of rationally engineered nanobodies with efficient neutralizing capacity against SARS-CoV-2 virus and resilience against mutational escape. In this work, we combined atomistic simulations and conformational dynamics analysis with the ensemble-based mutational profiling of binding interactions for a diverse panel of SARS-CoV-2 spike complexes with nanobodies. Using this computational toolkit, we identified dynamic signatures and binding affinity fingerprints for the SARS-CoV-2 spike protein complexes with nanobodies Nb6 and Nb20, VHH E, a pair combination VHH E+U, a biparatopic nanobody VHH VE, and a combination of CC12.3 antibody and VHH V/W nanobodies. Through ensemble-based deep mutational profiling of stability and binding affinities, we identify critical hotspots and characterize molecular mechanisms of SARS-CoV-2 spike protein binding with single ultra-potent nanobodies, nanobody cocktails and biparatopic nanobodies. By quantifying dynamic and energetic determinants of the SARS-CoV-2 S binding with nanobodies, we also examine the effects of circulating variants and escaping mutations. We found that mutational escape mechanisms may be controlled through structurally and energetically adaptable binding hotspots located in the host receptor-accessible binding epitope that are dynamically coupled to the stability centers in the distant epitope targeted by VHH U/V/W nanobodies. The results of this study suggested a mechanism in which through cooperative dynamic changes, nanobody combinations and biparatopic nanobody can modulate the global protein response and induce the increased resilience to common escape mutants.

S100 is a family of over 20 structurally homologous, but functionally diverse regulatory (calcium/zinc)-binding proteins of vertebrates. The involvement of S100 proteins in numerous vital (patho)physiological processes is mediated by their interaction with various (intra/extra)cellular protein partners, including cell surface receptors. Furthermore, recent studies revealed the ability of specific S100 proteins to affect cell signaling via direct interaction with cytokines. Previously, we have revealed binding of ca. 71% of the four-helical cytokines by S100P protein due to the presence in its molecule of a cytokine-binding site, which overlaps with the binding site for S100P receptor. Here we show that another S100 protein, S100A6 (pairwise sequence identity with S100P of 35%), specifically binds numerous four-helical cytokines. We have studied affinity of recombinant forms of 35 human four-helical cytokines covering all structural families of this fold to Ca2+-loaded recombinant human S100A6, using surface plasmon resonance spectroscopy. S100A6 recognizes 26 of the cytokines from all families of this fold with the equilibrium dissociation constants ranging from 0.3 nM to 12 µM. Overall, S100A6 interacts with ca. 73% of the four-helical cytokines studied to date with selectivity equivalent to that for S100P protein, with the differences limited to binding of Interleukin-2 and Oncostatin-M. The molecular docking study evidences presence in S100A6 molecule of a cytokine-binding site, analogous to that found in S100P. The findings argue the presence in some of the promiscuous members of S100 family of a site specific to a wide range of the four-helical cytokines. This unique feature of the S100 proteins potentially allows them to serve as universal inhibitors of signaling of the four-helical cytokines, which could be of value for reduction of severity of the disorders accompanied by excessive release of the cytokines.

The main objective of this study is to compare and investigate the proximate, sensory parameters of the biscuits fortified with (1) whey protein concentrate (WPC), (2) isolated soy protein (ISP) at various proportions as a source of protein enrichment. The fortified biscuits were analysed for sensory and proximate analysis. The study evaluated and compared the effect of fortifying whey protein concentrate and isolated soy protein at various proportions T0 control (0%), T1 (5%), T2 (10%), T3 (15%), T4 (20%). The protein content in both WPC and ISP samples tends to increase, but much higher in WPC formulated biscuits. At 20% WPC the protein content peaked to 29.11%, other nutritional components such as fat, ash, fibre and moisture content of WPC samples increased significantly, whereas no such significant changes were observed and only linear increase of protein content were in ISP fortified samples. The sensory analysis of ISP fortified samples were acceptable up to 10% proportion, exceeding 10% showed unacceptable results, on comparison WPC biscuits were acceptable at all proportions, more desirable at 20% WPC. Conclusively, fortifying biscuits with either WPC or ISP might be a source of protein without any significant effect on the quality of biscuits.

Background: Night-migratory birds sense the Earth´s magnetic field by an unknown molecular mechanism. Theoretical and experimental evidence support the hypothesis that light-induced formation of a radical-pair in European robin cryptochrome 4a, ErCry4a, is the primary signalling step in the retina of the bird. In the present work, we investigated a possible route of cryptochrome signalling involving the α-subunit of the cone specific heterotrimeric G protein from European robin. Methods: Protein-protein interaction studies include surface plasmon resonance, pulldown affinity binding and Förster resonance energy transfer. Results: Surface plasmon resonance studies showed direct interaction revealing high to moderate affinity for binding of non-myristoylated and myristoylated G protein to ErCry4a, respectively. Pulldown affinity experiments confirmed this complex formation in solution. We validated these in vitro data by monitoring the interaction between ErCry4a and G protein in a transiently transfected neuroretinal cell line using Förster resonance energy transfer. Conclusions: Our results suggest that ErCry4a and the G protein also interact in vivo and might constitute the first biochemical signalling step in radical-pair-based magnetoreception.

Obesity-associated metabolic abnormalities comprise of a cluster of conditions including dyslipidemia, insulin resistance, diabetes, and cardiovascular diseases that has affected more than 650 million people all over the globe. Obesity results from accumulation of white adipose tissues mainly due to the chronic imbalance of energy intake and energy expenditure. Variety of approaches to treat or prevent obesity, including lifestyle interventions, surgical weight loss procedures and pharmacological approaches to reduce energy intake and increase energy expenditure have failed to substantially decrease the prevalence of obesity. Brown adipose tissue (BAT), the primary source of thermogenesis in infants and small mammals may represent a promising therapeutic target to treat obesity by promoting energy expenditure through non-shivering thermogenesis mediated by mitochondrial uncoupling protein 1 (UCP1). Since the confirmation of functional BAT in adult humans by several groups, approximately a decade ago and its association with a favorable metabolic phenotype, intense interest on the significance of BAT in adult human physiology and metabolic health has emerged within the scientific community to explore its therapeutic potential for the treatment of obesity and metabolic diseases. Substantially decreased BAT activity in individuals with obesity indicates a role for BAT in setting of human obesity. On the other hand, BAT mass and its prevalence has been reported to correlate with lower body mass index (BMI), decreased age and glucose levels, leading to lower incidence of cardio metabolic diseases. Increased cold exposure in adult humans with undetectable BAT was associated with decreased body fat mass and increased insulin sensitivity. Deeper understanding of the role of BAT in human metabolic health and its inter-relationship with body fat distribution and deciphering proper strategies to increase energy expenditure by either increasing functional BAT mass, or inducing white adipose browning holds the promise for possible therapeutic avenues for the treatment of obesity and associated metabolic disorders.

The study presents a mathematical modeling for supporting the premise that our body is composed of blockchain systems. A cell population is considered a space with peer-to-peer communication networks for applying blockchain protocol. Transaction is defined as gene expression that constantly occurs for protein synthesis in each cell. The transaction process proceeds according to the blockchain protocol with sharing and recording the data on the blockchain ledger. The premise comes from the clues of the previous research such that the cell population is a complex structured network system having cell-to-cell connections. Although individual cells exhibit stochastic nonlinear dynamic behavior, cell population shows consensus behavior that reaches to ensemble through interaction among cells. It is inferred that gene expression is not regulated by the corresponding cell only nor determined by external intelligence such as the brain. This is achieved through a consensus through stochastic interactions between cells over the whole cell population. These findings imply that mathematical blockchain modeling is a suitable for gene expression process. The original contribution of the study is a methodology for applying mathematically the blockchain protocol to the real biological gene expression process. In other words, the DNA sequence is converted into a numeral bit sequence that makes it possible to implement the blockchain protocol. A new DNA sequence scheme is proposed with adding methylated cytosine and adenine as the 5th and 6th bases for including epigenetic information which has profound effect on gene expression and regulation. The methodology was applied to the real biological synthesis process of protein samples. The protein is composed of amino acids that are encoded by triplet codons of 216 kinds with 6 base RNA sequence. The gene expression information is traced backward from a synthesized protein sample, amino acids of codons, RNA transcript up to DNA sequence. One of the results is a numeric value in the form of a bit sequence with which mathematical blockchain modeling is applicable. The cryptographic authentication and the consensus process are mathematically proven to work properly by the blockchain protocol. It implies that the same protein is synthesized, but with different epigenetic data, then protein's latent material properties will certainly be different. Although the result has not been justified by the biologic experiment yet, it is sure that the biological hidden algorithm inside DNA sequence will be revealed by the binary bit-logic with physical on/off states which is mathematically proven. The research will greatly contribute to disease treatment and medicine development as well as epigenetics in the future.