Great strides in the field of lipidomics driven by advances in mass spectrometry techniques in the last decade have moved lipid analysis to a new level and significantly improved our understanding of lipid biochemistry. Multiple stage mass spectrometry (MSn) with high resolution mass spectrometry (HRMS) that allows sequential isolation, fragmentation, and recognition of ion structures, is a powerful tool for characterization of complex and diversified lipid in bacterial cells, in which lipids are often critical for cell aggregation and dissociation, and play important biological roles. In addition to common phospholipids, many bacteria contain unique lipids that are specific to the bacterium genus and even to the bacterium species. In this review, application of linear ion-trap (LIT) MSn in the structural characterization of native bacterial lipids including (1) novel lipids consisting of many isomeric structures, (2) lipids with unique functional groups and modification, (3) complex sphingolipids, peptidolipids, and lipocyclopeptides from various bacteria are presented. LIT MSn approach affords realization of the mechanisms underlying the fragmentation processes, resulting in identification of complex lipid structures that would be very difficult to define using other analytical methods.

A therapeutic system with the ability to target more than one protein is an important aim of cancer therapy since tumor growth is accompanied by dysregulation of many biological pathways. G-quadruplexes (G4s) are non-canonical structures formed by guanine-rich DNA or RNA oligonucleotides, with the ability to bind to different targets. In this study, we constructed ten novel bispecific G-quadruplex conjugates based on AT11, TBA, T40214 and T40231 aptamer structures, with the ability to bind two different targets at once in cancer cells. We analyzed the physicochemical aspects and the anticancer properties of novel molecules relating them with the single G-quadruplex unit and attempted to comprehend the correlation between the structures of bispecific G-quadruplexes with their biological activity. Our studies uncovered conjugates with considerable antiproliferative potential in HeLa and MCF-7 cancer cell lines, however with relatively low thermal stability or low nuclease resistance. Three conjugates among all studied oligonucleotides possess improved antiproliferative activity in MCF-7 cell line in comparison to their single G-quadruplex units leading to up to 90% inhibition of cancer cells growth, but their inhibitory potential is rather comparable to the effect observed for mix of two separate G-quadruplex units. Importantly, the conjugation enhances oligonucleotides enzymatic stability leading to the improvement of their therapeutic profile. The comprehensive studies presented herein indicate new approach for possibly effective cancer therapy and for the design of G4-based drugs.

Potato virus Y (PVY) is one of the most dangerous agricultural pathogens that causes substantial harm to vegetative propagated crops, such as potatoes (Solanum tuberosum L.). A necessary condition for PVY infection is an interaction between the plant cap-binding translation initiation factors eIF4E and a viral protein VPg, which mimics the cap-structure. In this study, we identified the point mutations in potato eIF4E1 and eIF4E2 that disrupt VPg binding while preserving the functional activity. For the structural interpretation of the obtained results, molecular models of all the studied forms of eIF4E1 and eIF4E2 were constructed and analyzed via molecular dynamics. The results of molecular dynamics simulations corresponds to the biochemical results and suggests that the β1β2 loop plays a key role in the stabilization of both eIF4E-cap and eIF4E-VPg complexes.

The PAS (Per-ARNT-Sim) domain is a sensory protein regulatory module found in archaea, prokaryotes, and eukaryotes. Histidine and serine/threonine protein kinases, chemo- and photoreceptors, circadian rhythm regulators, ion channels, phosphodiesterases, and other cellular response regulators are among these proteins. Hik33 is a multifunctional sensory histidine kinase that is implicated in cyanobacterial responses to cold, salt, hyperosmotic, and oxidative stressors. The functional roles of individual Hik33 domains in signal transduction were investigated in this study. Synechocystis Hik33 deletion variants were developed, in which either both or a portion of the transmembrane domains and/or the PAS domain were deleted. Cold stress was applied to the mutant strains either under illumination or in the dark. The findings show that the transmembrane domains govern temperature responses, whereas PAS domain may be involved in regulation of downstream gene expression in light-dependent manner.

The RNA/DNA helicase senataxin (SETX) has been involved in multiple crucial processes related to genome expression and integrity such us transcription termination, the regulation of transcription-replication conflicts and the resolution of R-loops. SETX has been the focus of numerous studies since the discovery that mutations in its coding gene are the root cause of two different neurodegenerative diseases: Ataxia with Oculomotor Apraxia type 2 (AOA2) and a juvenile form of Amyotrophic Lateral Sclerosis (ALS4). A plethora of cellular phenotypes have been described as the result of SETX deficiency, yet the precise molecular function of SETX as well as the molecular pathways leading from SETX mutations to AOA2 and ALS4 pathologies have remained unclear. However, recent data have shed light onto the biochemical activities and biological roles of SETX, thus providing new clues to understand the molecular consequences of SETX mutation. In this review we summarize near two decades of scientific effort to elucidate SETX function, we discuss strengths and limitations of the approaches and models used thus far to investigate SETX-associated diseases and suggest new possible research avenues for the study of AOA2 and ALS4 pathogenesis.

I-motifs are non-canonical DNA structures consisting of two parallel strands held together by hemiprotonated cytosine-cytosine+ base pairs, which intercalate to form a ordered column of stacked base pairs. This unique structure covers potential relevance in various fields, including gene regulation and biotechnological applications. A unique structural feature of I-motifs (iM), is the presence of sugar-sugar interactions through their extremely narrow minor grooves. Consistently, oligonucleotides containing pentose derivatives such as ribose, 2'-deoxyribose, arabinose, and 2'-deoxy-2'-fluoroarabinose highlighted a very different attitude to fold into iM. On the other hand, there is significant attention focused on exploring sugar-modifications that can increase nucleic acids resistance to nuclease degradation, a crucial requirement for therapeutic applications. An interesting example, not addressed in the iM field yet, is represented by hexitol nucleic acid (HNA), a metabolically stable six-membered ring analogue compatible with A-like double helix formation. Herein, we selected two DNA C-rich Tetrahymena telomeric sequences whose tetrameric iMs were already resolved by NMR and we investigated the iM folding of related HNA and RNA oligonucleotides by circular dichroism, differential scanning calorimetry and NMR. The comparison of their behaviours vs the DNA counterparts provided interesting insights into the influence of the sugar on iM folding. In particular, ribose and hexitol prevented iM formation. However, by clustering the hexitol-containing residues at the 3'-end, it was possible to modulate the distribution of the different topological species described for the DNA iMs. These data open new avenues for the exploitation of sugar modifications for I-motif characterization and applications.

Long non-coding (lnc) RNAs, once considered as junk and useless, are now broadly recognized to have major functions in the cell. LncRNAs are defined as non-coding RNAs of more than 200 nucleotides, regulate all steps of gene expression. Their origin is diverse, they can arise from intronic, intergenic or overlapping region, in sense or antisense direction. LncRNAs are mainly described for their action on transcription, while their action at the translational level is more rarely cited. However, the bibliography in the field is more and more abundant. The present synopsis of lncRNAs involved in the control of translation reveals a wide field of regulation of gene expression, with at least nine distinct molecular mechanisms. Furthermore, it appears that all these lncRNAs are involved in various pathologies including cancer, cardiovascular and neurodegenerative diseases.

R-loops are atypical, three-stranded nucleic acid structures that contain a stretch of RNA:DNA hybrids and an unpaired, single stranded DNA loop. R-loops are physiological relevant and can act as regulators of gene expression, chromatin structure, DNA damage repair and DNA replication. However, unscheduled and persistent R-loops are mutagenic and can mediate replication-transcription conflicts, leading to DNA damage and genome instability if left unchecked. Detailed transcriptome analysis unveiled that 85% of the human genome, including repetitive regions, hold transcriptional activity. This anticipates that R-loops management plays a central role for the regulation and integrity of genomes. This function is expected to have a particular relevance for repetitive sequences that make up to 75% of the human genome. Here, we review the impact of R-loops on the function and stability of repetitive regions such as centromeres, telomeres, rDNA arrays, transposable elements and triplet repeat expansions and discuss their relevance for associated pathological conditions.

Connexins (Cxs) are a family of transmembrane proteins located in the plasma membrane of human cells, among which connexin 43 (Cx43) is abundantly expressed in various types of human cells. Cx43, encoded by the gap junction protein alpha 1 (GJA1) gene, assembles into a hexameric structure in the Golgi apparatus and translocates to the plasma membrane to form hemichannels (Hcs), which pair with those of the cells in contact with each other and form gap junction intercellular communication (GJIC). The role of Cx43 as a connexin localized at the plasma membrane to perform channel functions is well recognized in previous studies, but recent studies have found that it can also be localized in the nucleus, mitochondria, or present in extracellular vesicles (EVs) and tunneling nanotubes (TNTs). Cx43 in the nucleus is involved in gene transcription regulation, cytoskeleton formation, cell migration and adhesion. Cx43 in mitochondria is involved in mitochondrial respiration-related functions, and Cx43 in extracellular vesicles and tunneling nanotubes is involved in distant cellular information exchange. It is because of the diverse distribution of subcellular localization of Cx43 that it is possible to explore the corresponding functions by analyzing its localization. In this review, we summarize the important roles of Cx43 in disease development from the perspective of subcellular localization, and provide new ideas for Cx43 as a therapeutic target and the search for related pathological mechanisms.

Glioblastoma (GBM) is the most aggressive and frequent type of primary brain cancer in adult patients. One of the key molecular features associated with GBM pathogenesis is the dysfunction of PTEN oncosuppressor. In addition to PTEN gene, humans and several primates possess processed PTEN pseudogene (PTENP1) that gives rise to long non-coding RNA lncPTENP1-S. Regulation and functions of PTEN and PTENP1 are highly interconnected, however, the exact molecular mechanism of how these two genes affect each other remains unclear. Here, we analyzed the methylation level of the CpG islands (CpGIs) in the promoter regions of PTEN and PTENP1 in patient-derived GBM neurospheres. We found that increased PTEN methylation corelates with decreased PTEN mRNA level. Unexpectedly, we showed the opposite trend for PTENP1. Using targeted methylation and demethylation of PTENP1 CpGI, we demonstrated that DNA methylation increases lncPTENP1-S expression in the presence of wild type PTEN protein but decreases lncPTENP1-S expression if PTEN protein is absent. Further experiments revealed that PTEN protein binds to PTENP1 promoter region and inhibits lncPTENP1-S expression if its CpGI is demethylated. Interestingly, we did not detect any effect of lncPTENP1-S on the level of PTEN mRNA, indicating that in GBM cells PTENP1 is a downstream target of PTEN rather than its upstream regulator. Finally, we studied the functions of lncPTENP1-S and demonstrated that it plays a pro-oncogenic role in GBM cells by upregulating the expression of cancer stem cell markers and decreasing cell adhesion.

Lysophospholipid acyltransferases (LPLATs), in concert with glycerol-3-phosphate acyltransferases (GPATs) and phospholipase A1/2s, orchestrate the compositional diversity of the fatty chains in membrane phospholipids. Fourteen LPLAT enzymes which come from two distinct families, AGPAT and MBOAT, have been identified, and in this mini-review we provide an overview of their roles in de novo and remodeling pathways of membrane phospholipid biosynthesis. Recently new nomenclature for LPLATs has been introduced (LPLATx, where x is a number 1-14), and we also give an overview of key biological functions that have been discovered for LPLAT1-14, revealed primarily through studies of LPLAT-gene-deficient mice as well as by linkages to various human diseases.

Melatonin is a pleiotropic neurohormone found in different animal, plant, and microorganism species. It is a product resulting from tryptophan metabolism in the pineal gland and is widely known for its ability to synchronize the circadian rhythm to antitumor functions in different types of cancers. The molecular mechanisms responsible for its immunomodulatory, antioxidant and cytoprotective effects involve binding to high-affinity G protein-coupled receptors and interactions with intracellular targets that modulate signal transduction pathways. In vitro and in vivo studies have reported the therapeutic potential of melatonin in different infectious and parasitic diseases. In this review, the protective and pathophysiological roles of melatonin in fighting protozoan and helminth infections and the possible mechanisms involved against these stressors will be discussed.

Non ketotic hyperglycinemia (NKH) is an inborn error of glycine metabolism caused by mutations in the genes encoding glycine cleavage system proteins. Classic NKH has a neonatal onset, and patients present with severe neurodegeneration. Although glycine accumulation has been implicated in NKH pathophysiology, the exact mechanisms underlying the neurological damage and white matter alterations remain unclear. We investigated the effects of glycine in the brain of neonatal rats and MO3.13 oligodendroglial cells. Glycine decreased myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) in the corpus callosum and striatum of rats on post-natal day (PND) 15. Glycine also reduced neuroglycan 2 (NG2) and N-methyl-d-aspartate receptor subunit 1 (NR1) in the cerebral cortex and striatum on PND15. Moreover, glycine reduced striatal glutamate aspartate transporter 1 (GLAST) content and neuronal nucleus (NeuN), and increased glial fibrillary acidic protein (GFAP) on PND15. Glycine also increased DCFH oxidation and malondialdehyde levels and decreased GSH concentrations in the cerebral cortex and striatum on PND6, but not on PND15. Glycine further reduced viability but did not alter DCFH oxidation and GSH levels in MO3.13 cells after 48- and 72-h incubation. These data indicate that impairment of myelin structure and glutamatergic system and induction of oxidative stress are involved in the neuropathophysiology of NKH.

Viral nanoparticles (VNPs) are self-assembling, adaptable delivery systems for vaccines and other therapeutic agents used in a variety of biomedical applications. The potential of viruses to invade and infect various hosts and cells renders them suitable as potential nanocarriers, possessing distinct functional characteristics, immunogenic properties, and improved biocompatibility and biodegradability. VPNs are frequently produced through precise genetic or chemical engineering, which involves adding diverse sequences or functional payloads to the capsid protein (CP). Several spherical and helical plant viruses, bacteriophages, and animal viruses are currently being used as VNPs, or non-infectious virus-like particles (VLPs). In addition to their broad use in cancer therapy, vaccine technology, diagnostics, and molecular imaging, VNPs have made important strides in the realms of tissue engineering, biosensing, and antimicrobial prophylaxis. They are also being used in energy storage cells due to their binding and piezoelectric properties. The large-scale production of VNPs for research, preclinical testing, and clinical use is fraught with difficulties, such as those relating to cost-effectiveness, scalability, and purity. Consequently, many plants- and microorganism-based platforms are being developed, and newer viruses are being explored. The goal of the current review is to provide an overview of these advances.

The human telomere oligonucleotide, d[TAGGG(TTAGGG)2TTAGG] (TAGGG), can adopt two distinct 2-G-quartet G-quadruplex structures at pH 7.0 and 5.0, referred to as the TD and KDH+ forms, respectively. By using a combination of NMR and computational techniques, we determined high-resolution structures of both forms, which revealed unique loop architectures, base triples, and base pairs that play a crucial role in the pH-driven structural transformation of TAGGG. Our study demonstrated that TAGGG represents a reversible pH-driven switch system where the stability and pH-induced structural transformation of the G-quadruplexes are influenced by the terminal residues and base triples. Gaining insight into the factors that regulate the formation of G-quadruplexes and their pH-sensitive structural equilibrium holds great potential for the rational design of novel DNA based pH-driven switches. These advancements in understanding create exciting opportunities for applications in the field of nanotechnology, specifically in the development of bio-nano-motors.

Mammalian Base Excision Repair (BER) DNA ligases I and IIIα (LigI, LigIIIα) are major determinants of DNA repair fidelity, alongside with DNA polymerases. Here we compared activities of human LigI and LigIIIα on specific and nonspecific substrates representing intermediates of distinct BER sub-pathways. The enzymes differently discriminate mismatches in the nicked DNA, depending on their identity and position, but are both more selective against the 3'-end non-complementarity. LigIIIα is less active than LigI in premature ligation of one-nucleotide gapped DNA and more efficiently discriminates misinsertion products of DNA polymerase β-catalyzed gap filling, that reinforces a leading role of LigIIIα in the accuracy of short-patch BER. LigI and LigIIIα reseal the intermediate of long-patch BER containing an incised synthetic AP site (F) with different efficiencies, depending on the DNA sequence context, 3'-end mismatch presence and coupling of the ligation reaction with DNA repair synthesis. Processing of this intermediate in the absence of flap endonuclease 1 generates non-canonical DNAs with bulged F site, which are very inefficiently repaired by AP endonuclease 1 and represent potential mutagenic repair products. The extent of conversion of the 5'-adenylated intermediates of specific and nonspecific substrates is revealed to depend on the DNA sequence context; a higher sensitivity of LigI to the sequence is in line with the enzyme structural feature of DNA binding. LigIIIα exceeds LigI in generation of potential abortive ligation products, justifying importance of XRCC1-mediated coordination of LigIIIα and aprataxin activities for the efficient DNA repair.