Cancer has high mortality rate and occupies second position among major diseases. Despite extensive research and therapies, in every nook and corner of the world, death rate is increasing exponentially. Hallmarks of cancer are benchmarks of cancer cells describing the fundamental principle and capabilities of the cells transforming from normal to malignant tumour. One of the major ones among them is the deregulation of cellular metabolism or metabolic reprogramming, involving alterations in glucose and lipid metabolism. Progressive research in this area has visualized the vital role of lncRNAs in lipid metabolism with respect to AML. lncRNAs involve in various cellular processes and also contribute for significant functions of the cell like chromatin remodelling, transcriptional activation and repression, gene regulation, immune response, cell differentiation, and cell cycle regulation, in addition to oncogenic processes such as proliferation, angiogenesis, migration, and apoptosis. Structural similarities are observed among mRNAs and lncRNAs in terms of poly A-tail and 5' cap however protein-coding regions are lacking. A large body of evidence has shown that lncRNAs directly or indirectly mediate lipid metabolism by activating downstream genes. Considering their potential involvement in leukemia, these lncRNAs can be explored and considered as biomarkers for therapeutics, prognosis, and diagnosis. The present review is planned to summarize the functional classification of lncRNAs, the role of lipid metabolism in cancer, different lncRNAs involved in leukemia, and different cancer types related to lipid metabolism.

Kawasaki disease (KD) is an acute vasculitis primarily affecting children, with a potential risk of developing coronary artery aneurysms (CAAs) and cardiovascular complications. The emergence of non-coding RNAs (ncRNAs), including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), has provided insights into Kawasaki disease pathogenesis and opened new avenues for diagnosis and therapeutic intervention. Furthermore, polymorphism analysis of ncRNA genes offers significant insights into genetic predisposition to Kawasaki disease, facilitating tailored treatment approaches and risk assessment to improve patient outcomes. Exosomal ncRNAs, which are ncRNAs encapsulated within extracellular vesicles, have garnered significant attention as potential biomarkers for Kawasaki disease and CAA due to their stability and accessibility in biological fluids. This review comprehensively discusses the biogenesis, components, and potential of exosomal and non-exosomal ncRNAs in Kawasaki disease diagnosis and prognosis prediction. It also highlights the roles of non-exosomal ncRNAs, such as miRNAs, lncRNAs, and circRNAs, in Kawasaki disease pathogenesis and their implications as therapeutic targets. Additionally, the review explores the current diagnostic and therapeutic approaches for Kawasaki disease and emphasizes the need for further research to validate these ncRNA-based biomarkers in diverse populations and clinical settings.

RNA targeting represents an original and promising approach to the discovery of new therapeutic tools against numerous diseases. The majority of intracellular RNAs are noncoding RNAs that play key regulatory functions in many physiological processes. Among these RNAs, long noncoding RNAs (lncRNAs) constitute the largest class of noncoding transcripts and have been shown to play important functional roles in development and disease processes. In this work, we developed a set of biochemical assays for the discovery of efficient small-molecule lncRNA ligands selective for their target, focusing on MALAT1 lncRNA. The latter bears a particular structure including a triple helical region important for its function, and it has been linked to cancer cells' proliferation. However, its role in cancer still needs to be completely elucidated. The application of these assays to an original library of RNA binders allowed for the discovery of unprecedented ligands of the MALAT1 triple helix able to inhibit and destabilize the triple helical MALAT1 structure. The set of screening and validation assays developed could find application in the discovery of new MALAT1 binders, and the new chemical scaffolds discovered in this study represent promising chemical probes for the study of the biological role of MALAT1 in disease.

The sea louse, Caligus rogercresseyi, is one of the main concerns in the Chilean salmon industry. The free-living copepodid stage can recognize the host and initiate the parasitic phase, where environmental factors can shape the host recognition process. This study aimed to explore the ecological influence on the transcriptome of copepodids infesting Atlantic salmon experimentally exposed to different salinity and temperature (S/T) conditions. Herein, 200 salmon were infested with 35 copepodids per fish previously acclimatized to four S/T treatments: 32 and 26 PSU; 8 and 16°C. After 48 h of infestation, the attached copepodids from each experimental group were counted and digitalized for geometric morphometric analysis. Copepodids were then collected for RNA sequencing to analyze transcriptome modulation and gene regulation. Morphological changes in copepodids were mainly associated with temperature rather than salinity conditions. The transcriptome survey revealed molecular signatures related to salinity and temperature changes, where salinity drives the gene expression of copepodids. Notably, specific genes, such as those encoding cuticle proteins and trypsin-like kinases, were regulated by all three post-transcriptional mechanisms assessed: alternative splicing, miRNA, and gene fusion. The transcriptome analysis revealed that trypsin-like kinase genes exhibited upregulation and downregulation across the various S/T conditions. In contrast, cuticle protein genes were consistently downregulated in the 32 PSU/8°C, 26 PSU/8°C, and 26 PSU/16°C groups compared to the 32 PSU/16°C control. This suggests that the three post-transcriptional mechanisms may exert a combined influence on the expression of specific genes, potentially driven by salinity and temperature environmental conditions in sea lice biology.

Transcriptional adaptation (TA) is a cellular process whereby mRNA-destabilizing mutations are associated with the transcriptional upregulation of so-called adapting genes. The nature of the TA-triggering factor(s) remains unclear, namely whether an mRNA-borne premature termination codon or the subsequent mRNA decay process, and/or its products, elicits TA. Here, working with mouse Actg1, we first establish two types of perturbations that lead to mRNA destabilization: Cas9-induced mutations predicted to lead to mutant mRNA decay, and Cas13d-mediated mRNA cleavage. We find that both types of perturbations are effective in degrading Actg1 mRNA, and that they both upregulate Actg2. Notably, increased chromatin accessibility at the Actg2 locus was observed only in the Cas9-induced mutant cells but not in the Cas13d-targeted cells, suggesting that chromatin remodeling is not required for Actg2 upregulation. We further show that ribozyme-mediated Actg1 pre-mRNA cleavage also leads to a robust upregulation of Actg2, and that this upregulation is again independent of chromatin remodeling. Together, these data highlight the critical role of RNA destabilization events as a trigger for TA, or at least a TA-like response.

Ovarian cancer remains a major challenge in oncology due to its complex biology and late-stage diagnosis. Recent advances in molecular biology have highlighted the crucial role of non-coding RNAs (ncRNAs) in regulating apoptosis and cancer progression. NcRNAs, including microRNAs, long non-coding RNAs, and circular RNAs, have emerged as significant players in the molecular networks governing ovarian cancer. Despite these insights, the precise mechanisms by which ncRNAs influence ovarian cancer pathology are not fully understood. This complexity, combined with the heterogeneity of the disease and the development of treatment resistance, poses substantial obstacles to effective therapeutic development. Additionally, the lack of reliable early detection methods further complicates treatment strategies. This manuscript reviews the current state of research on ncRNAs in ovarian cancer, discusses the challenges in translating these findings into clinical applications, and outlines potential future directions. Emphasis is placed on the need for integrated approaches to unravel the intricate roles of ncRNAs, improve early detection, and develop personalized treatment strategies to address the diverse and evolving nature of ovarian cancer. While these findings provide valuable insights, it is crucial to recognize that many results are based on preclinical studies and require further validation to establish their clinical applicability.

Cancer is a rising issue worldwide, and numerous studies have focused on understanding the underlying reasons for its occurrence and finding proper ways to defeat it. By applying technological advances, researchers are continuously uncovering and updating treatments in cancer therapy. Their vast functions in the regulation of cell growth and proliferation and their significant role in the progression of diseases, including cancer. This review provides a comprehensive analysis of ncRNAs in breast cancer, focusing on long non-coding RNAs such as HOTAIR, MALAT1, and NEAT1, as well as microRNAs such as miR-21, miR-221/222, and miR-155. These ncRNAs are pivotal in regulating cell proliferation, metastasis, drug resistance, and apoptosis. Additionally, we discuss experimental approaches that are useful for studying them and highlight the advantages and challenges of each method. We then explain the results of these clinical trials and offer insights for future studies by discussing major existing gaps. On the basis of an extensive number of studies, this review provides valuable insights into the potential of ncRNAs in cancer therapy. Key findings show that even though the functions of ncRNAs are vast and undeniable in cancer, there are still complications associated with their therapeutic use. Moreover, there is an absence of sufficient experiments regarding their application in mouse models, which is an area to work on. By emphasizing the crucial role of ncRNAs, this review underscores the need for innovative approaches and further studies to explore their potential in cancer therapy.

Autoimmune diseases (ADs) are a group of complex, chronic conditions characterized by disturbance of immune tolerance, with examples including systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, and psoriasis. These diseases have unclear pathogenesis, and traditional therapeutic approaches remain limited. However, advances in high-throughput histology technology and scientific discoveries have led to the identification of various pathogenic factors contributing to ADs. Coupled with improvements in RNA nucleic acid-based drug synthesis, design, and delivery, RNA-based therapies have been extensively investigated for their potential in treating ADs. This paper reviews the progress in the use of miRNAs, lncRNAs, circRNAs, siRNAs, antisense oligonucleotides (ASOs), aptamers, mRNAs, and other RNA-based therapies in ADs, focusing on their therapeutic potential and application prospects, providing insights for future research and clinical treatment of autoimmune diseases.

The occurrence of diabetic kidney disease (DKD), a critical microvascular issue in diabetes, is progressively on the rise. In recent years, long noncoding RNAs (lncRNAs) have garnered considerable attention as a novel and critical layer of biological regulation. Our knowledge regarding the roles and underlying mechanisms of lncRNAs in various diseases, including DKD, continues to evolve. Similarly, microRNAs (miRNAs), which are small noncoding RNAs, have been recognized as crucial contributors to cellular processes and disease pathogenesis. Emerging studies have highlighted the complex interactions between lncRNAs and miRNAs, particularly in the context of DKD, underscoring their importance in complex human diseases. Renal intrinsic cell damage is an important cause of inducing DKD. Persistent high glucose stimulation leads to remodeling of renal intrinsic cells and a cascade of pathological changes. This article aims to review recent literature on the lncRNAs-mediated regulation of miRNAs affecting renal intrinsic cells in DKD and to propose novel molecular-level therapeutic strategies for DKD. Through in-depth investigation of this dynamic molecular interaction, we can gain a profound understanding of the potential mechanisms underlying diabetic nephropathy, potentially identifying new targets for therapeutic intervention and paving the way for personalized and effective treatments.

Microscopy approaches are frequently used to decipher the localization and quantify the abundance of biologically relevant molecular targets within single cells. Recent research has applied many optical imaging techniques to specifically visualize epigenetic modifications, the mechanisms by which organisms control gene expression in response to environmental factors. While many molecular and omics-based approaches are used to understand epigenetic mechanisms, imaging approaches provide spatial information that supplies greater context for discerning function. Thus, labeling approaches have been developed to quantify and visualize epigenetic targets using various fluorescence microscopy, electron microscopy, and super-resolution microscopy techniques. Here, we synthesize information about microscopy methods that enable visualization of epigenetic marks including DNA methylation, histone modifications, and localization of RNAs, which provide insights into mechanisms involved in chromatin remodeling and gene expression. The ability to determine how and where specific epigenetic marks manifest structurally and functionally in cells demonstrates the power of microscopy in aiding our understanding of epigenetic processes.

Fluorescent RNAs (FRs), which are RNA aptamers that bind and activate their cognate small fluorogenic dyes, have provided a particularly useful approach for imaging RNAs in live cells. Although the color palette of FRs is greatly expanded, a bright and stable cyan FR with good biocompatibility and biorthogonality with currently available FRs remains desirable but is not yet developed. Herein, the development of Myosotis is described, an RNA aptamer that emits bright cyan fluorescence upon binding a novel GFP chromophore-like fluorophore called DBT. Myosotis has a nanomolar affinity for DBT and shows a weak dependence on magnesium for folding. Further studies reveal that the Myosotis-DBT complex has a long fluorescence lifetime, good photostability, and enhance cellular brightness. It is further shown that Myosotis-DBT is biorthogonal to Pepper and Clivia FRs, allowing multiplex fluorescence imaging of RNA in live bacteria. Myosotis can also use to image mRNA in live bacteria, revealing potential coupling between mRNA translation and stability. It is believed that this cyan FR will be a useful tool for studying the functionality and mechanism of RNA underlying diverse biological processes.

The fact that protein universe is enriched in intrinsic disorder is an accepted truism now. It is also recognized that the phenomenon of protein intrinsic disorder contains keys to answer numerous questions that do not have obvious solutions within the classic "lock-and-key"-based structure-function paradigm. In fact, reality is much more complex than the traditional "one-gene - one-protein - one-function" model, as many (if not most) proteins are multifunctional. This multifunctionality is commonly rooted in the presence of the intrinsically disordered or structurally flexible regions in a protein. Here, in addition to various events at the DNA (genetic variations), mRNA (alternative splicing, alternative promoter usage, alternative initiation of translation, and mRNA editing), and protein levels (post-translational modifications), intrinsic disorder and protein functionality are crucial for generation of proteoforms, which are functionally and structurally different protein forms produced from a single gene. Therefore, since a given protein exists as a dynamic conformational ensemble containing multiple proteoforms characterized by a broad spectrum of structural features and possessing various functional potentials, "protein structure-function continuum" model represents a more realistic way to correlate protein structure and function.

Humans have more than 270,000 lncRNAs. Among these, lncRNA HOXA-AS2 is considered a transformative gene involved in various cellular processes, including cell proliferation, apoptosis, migration, and invasion. Thus, it can be regarded as a potential tumor marker for both diagnosis and prognosis. Aberrant expression of lncRNAs is associated with many cancers, including hepatocellular carcinoma (HCC), gallbladder carcinoma (GBC), acute promyelocytic leukemia (APL), lung cancer (LC), prostate cancer (PC), osteosarcoma (OS), colorectal cancer (CRC), cervical cancer (CC), and acute myeloid leukemia (AML). Targeting lncRNAs could be a promising strategy to complement or replace current cancer treatments. As a non-coding oncogene, lncRNA HOXA-AS2 is implicated in multiple cancers and could serve as a potential biomarker for various malignancies. The tumor size and disease stage of several cancers are correlated with HOXA-AS2 expression. Silencing HOXA-AS2 effectively suppresses tumor cell proliferation and promotes apoptosis, thereby inhibiting the progression of multiple cancer types. The regulatory mechanisms of HOXA-AS2 include inducing epithelial-mesenchymal transition (EMT), overexpressing B-cell lymphoma-2 (Bcl-2) and MYC proto-oncogene (c-Myc), gene silencing, activating AKT-MMP signaling pathways, EZH2 and LSD1, and functioning within a competing endogenous RNA (ceRNA) regulatory network by competitively binding miRNAs. This review surveys recent research on the structure, biological functions, abnormal expression, regulatory mechanisms, and diagnostic and therapeutic potential of HOXA-AS2 in various cancers.

Long non-coding RNAs (lncRNAs), serving as diverse functional regulators, are abundantly expressed in the testis. However, many testis-specific or preferentially expressed lncRNAs remain uncharacterized. Here, we report a testis-specific lncRNA, 1700052I22Rik, which exhibits a dynamic expression pattern during spermatogenesis. Our findings demonstrate that knockout of 1700052I22Rik in mice leads to reduced sperm counts and subfertility in males, as well as defective spermatid chromatin condensation. We further elucidate the underlying mechanism by which 1700052I22Rik modulates the translation of protamine 1 (PRM1) through interaction with Y-box binding protein 2 (YBX2). Collectively, our results uncover a crucial role for the testis-specific lncRNA 1700052I22Rik in regulating spermatid chromatin condensation in mice, providing novel insights into the functions of lncRNAs in spermatogenesis and potential targets for the diagnosis and treatment of male infertility.

Epigenetics is an important source of variation in complex traits that is not due to changes in DNA sequences, and is dependent on the environment the individuals are exposed to. Therefore, we aimed to estimate transgenerational epigenetic heritability, percentage of resetting epigenetic marks, genetic parameters, and predicting breeding values using genetic and epigenetic models for growth, body composition, and reproductive traits in Landrace pigs using routinely recorded datasets. Birth and weaning weight, backfat thickness, total number of piglets born, and number of piglets born alive (BW, WW, BF, TNB, and NBA, respectively) were investigated. Models including epigenetic effects had a similar or better fit than solely genetic models. Including genomic information in epigenetic models resulted in large changes in the variance component estimates. Transgenerational epigenetic heritability estimates ranged between 0.042 (NBA) to 0.336 (BF). The reset coefficient estimates for epigenetic marks were between 80% and 90%. Heritability estimates for the direct additive and maternal genetic effects ranged between 0.040 (BW) to 0.502 (BF) and 0.034 (BF) to 0.134 (BW), respectively. Repeatability of the reproductive traits ranged between 0.098 (NBA) to 0.148 (TNB). Prediction accuracies, bias, and dispersion of breeding values ranged between 0.199 (BW) to 0.443 (BF), -0.080 (WW) to 0.034 (NBA), and -0.134 (WW) to 0.131 (TNB), respectively, with no substantial differences between genetic and epigenetic models. Transgenerational epigenetic heritability estimates are moderate for growth and body composition and low for reproductive traits in North American Landrace pigs. Fitting epigenetic effects in genetic models did not impact the prediction of breeding values.

Bacteriophages, or phages, depend on their bacterial hosts for proliferation, leading to a coevolutionary relationship characterized by on-going arms races, where bacteria evolve diverse antiphage defense systems. The development of in silico methods and high-throughput screening techniques has dramatically expanded our understanding of bacterial antiphage defense systems, enormously increasing the known repertoire of the distinct mechanisms across various bacterial species. These advances have revealed that bacterial antiphage defense systems exhibit a remarkable level of complexity, ranging from highly conserved to specialized mechanisms, underscoring the intricate nature of bacterial antiphage defense systems. In this review, we provide a concise snapshot of antiphage defense research highlighting two preponderantly commandeered approaches and classification of the known antiphage defense systems. A special focus is placed on the model bacterial pathogen, Pseudomonas aeruginosa in antiphage defense research. We explore the complexity and adaptability of these systems, which play crucial roles in genome evolution and adaptation of P. aeruginosa in response to an arsenal of diverse phage strains, emphasizing the importance of this organism as a key emerging model bacterium in recent antiphage defense research.

Cancer can take years to develop, both at its beginning and during its development. All typical epithelial cancers have a long latency period, sometimes 20 years or more, and if they are clinically detected, distinct genes may include infinite mutations. Long non-coding RNAs (LncRNAs) are a subset of RNAs that regulate many biological processes, including RNA processing, epigenetic control, and signal transduction. Current studies show that lncRNAs, which are dysregulated in cancer, play a significant function in the growth and spread of the illness. LncRNAs have been connected to the overexpression of specific proteins that function in tumors' spread and growth. Moreover, through translational inhibition, microRNAs (miRNAs) regulates gene expression sequence specifically. Apart from that, non-coding RNAs known as miRNAs, with a length of around 22 nucleotides, controls gene expressions in a sequence-specific way either by preventing translation or degrading messenger RNA (mRNA). Quercetin appears to have a significant role in altering miRNA and lncRNA expression, which is linked to variations in the production of oncogenes, tumor suppressors, and proteins produced from cancer. Quercetin may change the earliest epigenetic modifications related to cancer prevention in addition to its usual antioxidant or anti-inflammatory effects. It would be beneficial to have more in-depth information on how Quercetin modulates miRNAs and lncRNAs to use it as a cancer therapeutic strategy. Here, we go through what is known about Quercetin's potential to modulate miRNAs and lncRNAs in various malignancies.

Background/objectives: Methyl jasmonate is a plant signaling molecule involved in a wide range of functions, including stress responses. This study investigates the relative differential expression of microRNAs and their target genes in response to methyl jasmonate treatment of Scots pine needles. Methods: A combined strategy of high-throughput sequencing and in silico prediction of potential target genes was implemented. Results: a total of 58 differentially expressed (DE) microRNAs (miRNAs) (43 up-regulated and 15 down-regulated), belonging to 29 miRNA families, were identified. The 41 DE miRNAs from 17 families were conifer-specific miRNA families-miR946, miR947, miR950, miR1312, miR1313, miR1314, miR3693, miR3107, miR11452, miR11466, miR11487, miR11490, miR11504, miR11511, miR11532, miR11544, and miR11551. The other DE miRNAs (miR159, miR164, miR169, miR396, miR397, miR398, miR408, miR535) were conserved miRNAs, which are also found in angiosperm species. Transcriptome analysis identified 389 gene transcripts with 562 miRNA-target sites targeted by 57 of the 58 DE miRNAs. Of these, 250 target genes with 138 different GO annotations were found for the 41 DE conifer-specific conserved miRNAs. Conclusions: The 26 DE miRNAs from 14 DE miRNA families, of which almost all (12 families, 24 miRNAs) are conifer specific, and were associated with 68 disease resistance and TMV resistance proteins, TIR-NBS-LRR, LRR receptor-like serine/threonine-protein kinase, putative CC-NBS-LRR protein, and putative NBS-LRR protein target transcripts with 29 target gene GO term descriptions. Some of the genes targeted by conifer-specific miRNAs have been previously reported to be targeted by other miRNAs in angiosperms, indicating that the miRNA-target gene regulation system can vary between species.

lncRNAs (long non-coding RNAs) are heterogeneous RNA molecules that modulate various cellular processes, such as proliferation, differentiation, migration, invasion, and apoptosis, via different mechanisms. An increasing amount of research indicates that abnormal expression of lncRNA influences the development of drug resistance as well as the genesis and advancement of cancer, including melanoma. Furthermore, they are attractive biomarkers for non-invasive cancer diagnostics due to their strongly modulated expression and improved tissue and disease specificity. This review offers a succinct overview of the present understanding concerning the potential diagnostic biomarker potential of lncRNAs in melanoma. Cell death occurs frequently during growth and throughout life and is an active, organized, and genetically determined process. It is essential for the regulation of homeostasis. Controlled cell death and non-programmed cell death are both forms of cell death. The most prevalent forms of regulatory cell death are pyroptosis, ferroptosis, autophagy, necroptosis, necrosis, and apoptosis. Ferroptosis, pyroptosis, and autophagy are less common forms of cell death compared to necrosis, apoptosis, and necroptosis. ncRNAs are regulatory RNA molecules that are not involved in encoding proteins. They primarily consist of circular RNAs (circ RNAs), lncRNAs, and microRNAs (miRNAs). Moreover, non-coding RNAs have the ability to modulate tumor cell autophagy, pyroptosis, and ferroptosis at the transcriptional or post-transcriptional stage, as well as function as oncogenes and tumor suppressor genes, which can have considerable effects on the incidence and growth of tumors. This review concentrated on the recent advancements in the research of the diagnostic and therapeutic functions of ncRNAs in the regulation of programmed cell death in melanoma.

Pentatricopeptide repeat proteins (PPR) are a large family of modular RNA-binding proteins, whereby each module can be modified to bind to a specific ssRNA nucleobase. As such, there is interest in developing 'designer' PPRs (dPPRs) for a range of biotechnology applications, including diagnostics or in vivo localization of ssRNA species; however, the mechanistic details regarding how PPRs search for and bind to target sequences is unclear. To address this, we determined the structure of a dPPR bound to its target sequence and used two- and three-color single-molecule fluorescence resonance energy transfer to interrogate the mechanism of ssRNA binding to individual dPPRs in real time. We demonstrate that dPPRs are slower to bind longer ssRNA sequences (or could not bind at all) and that this is, in part, due to their propensity to form stable secondary structures that sequester the target sequence from dPPR. Importantly, dPPR binds only to its target sequence (i.e. it does not associate with non-target ssRNA sequences) and does not 'scan' longer ssRNA oligonucleotides for the target sequence. The kinetic constraints imposed by random 3D diffusion may explain the long-standing conundrum of why PPR proteins are abundant in organelles, but almost unknown outside them (i.e. in the cytosol and nucleus).

Recent studies confirm that nanoplastics (NP) cause severe microbial imbalances in various ecosystems, significantly affecting microbial diversity and abundance. Hydroponic systems vital for lettuce production are increasingly threatened by NP contamination in irrigation water and this issue is gaining global attention. This study investigates microbial species in hydroponic irrigation water altered by NP exposure and their impact on lettuce growth. While NP (108-1010 particles/L) did not directly harm or accumulate in lettuce, significant changes in water parameters and microbial communities were observed, particularly an increase in Curvibacter fontanus abundance. Inoculation of sterile irrigation water with NP and C. fontanus led to lettuce mortality, suggesting C. fontanus as a critical mediator. Furthermore, extracellular vesicles (EVs) isolated from C. fontanus, treated with NP, were shown to suppress leaf development, growth, antioxidant defenses, and lettuce survival. This study concludes that NP-induced microbial shifts, particularly involving C. fontanus EVs, indirectly harm hydroponic lettuce production.

Non-coding RNAs (ncRNAs), especially microRNAs (miRNAs) and long ncRNAs (lncRNAs), are essential regulators of processes, such as the cell cycle and apoptosis. In addition to interacting with intracellular complexes and participating in diverse molecular pathways, ncRNAs can be used as clinical diagnostic biomarkers and therapeutic targets for fighting cancer. Studying ncRNA gene clusters is crucial for understanding their role in cancer and developing new treatments. LOC124905135 is a protein-coding gene encoding a collagen alpha-1(III) chain-like protein, and also acts as a gene for several ncRNAs, including miR-3619, PRR34 antisense RNA 1 (PRR34-AS1), PRR34, long intergenic ncRNA 2939 (LINC02939), LOC112268288, and MIRLET7BHG. It also serves as a host gene for three miRNAs (hsa-let7-A3, hsa-miR-4763, and hsa-let-7b). Notably, the ncRNAs derived from this particular genomic region significantly affect various cell functions, including the cell cycle and apoptosis. This cluster of ncRNAs is dysregulated in several types of cancer, exhibiting mutations, alterations in copy number, and being subject to DNA methylation and histone modification. In summary, the ncRNAs derived from the LOC124905135 cluster could be used as targets for diagnosis, therapy monitoring, and drug discovery in human cancers.

RNA provides the genetic blueprint for many pathogenic viruses, including SARS-CoV-2. The propensity of RNA to fold into specific tertiary structures enables the biomolecular recognition of cavities and crevices suited for the binding of drug-like molecules. Despite increasing interest in RNA as a target for chemical biology and therapeutic applications, the development of molecules that recognize RNA with high affinity and specificity represents a significant challenge. Here, we report a strategy for the discovery and selection of RNA-targeted macrocyclic peptides derived from combinatorial libraries of peptide macrocycles displayed by bacteriophages. Specifically, a platform for phage display of macrocyclic organo-peptide hybrids (MOrPH-PhD) was combined with a diverse set of non-canonical amino acid-based cyclization modules to produce large libraries of 107 structurally diverse, genetically encoded peptide macrocycles. These libraries were panned against the -1 programmed ribosomal frameshifting stimulatory sequence (FSS) RNA pseudoknot of SARS-CoV-2, which revealed specific macrocyclic peptide sequences that bind this essential motif with high affinity and selectivity. Peptide binding localizes to the FSS dimerization loop based on chemical modification analysis and binding assays and the cyclic peptides show specificity toward the target RNA over unrelated RNA pseudoknots. This work introduces a novel system for the generation and high-throughput screening of topologically diverse cyclopeptide scaffolds (multiplexed MOrPH-PhD), and it provides a blueprint for the exploration and evolution of genetically encoded macrocyclic peptides that target specific RNAs.

RNA is a central molecule in life, involved in a plethora of biological processes and playing a key role in many diseases. Targeting RNA emerges as a significant endeavor in drug discovery, diverging from conventional protein-centric approaches to tackle various pathologies. Whilst identifying small molecules that bind to specific RNA regions is the first step, the abundance of non-functional RNA segments renders many interactions biologically inert. Consequently, small molecule binding does not necessarily meet stringent criteria for clinical translation, calling for solutions to push the field forward. Converting RNA-binders into RNA-degraders presents a promising avenue to enhance RNA-targeting. This mini-review outlines strategies and exemplars wherein simple small molecule RNA binders are reprogrammed into active degraders through the linkage of functional groups. These approaches encompass mechanisms that induce degradation via endogenous enzymes, termed RIBOTACs, as well as those with functional moieties acting autonomously to degrade RNA. Through this exploration, we aim to offer insights into advancing RNA-targeted therapeutic strategies.

RNA sequencing (RNA-seq) has undergone substantial advancements in recent decades and has emerged as a vital technique for profiling the transcriptome. The transition from bulk sequencing to single-cell and spatial approaches has facilitated the achievement of higher precision at cell resolution. It provides valuable biological knowledge about individual immune cells and aids in the discovery of the molecular mechanisms that contribute to the development of autoimmune diseases. Celiac disease (CeD) is an autoimmune disorder characterized by a strong immune response to gluten consumption. RNA-seq has led to significantly advanced research in multiple fields, particularly in CeD research. It has been instrumental in studies involving comparative transcriptomics, nutritional genomics and wheat research, cancer research in the context of CeD, genetic and noncoding RNA-mediated epigenetic insights, disease monitoring and biomarker discovery, regulation of mitochondrial functions, therapeutic target identification and drug mechanism of action, dietary factors, immune cell profiling and the immune landscape. This review offers a comprehensive examination of recent RNA-seq technology research in the field of CeD, highlighting future challenges and opportunities for its application.

Ribonucleic acid (RNA) undergoes dynamic changes in its structure and function under various intracellular and extracellular conditions over time. However, there is a lack of research on the concept of the RNA age to describe its diverse fates. This study proposes a definition of RNA age to address this issue. RNA age was defined as a sequence of numbers wherein the elements in the sequence were the nucleotide ages of the ribonucleotide residues in the RNA. Mean nucleotide age was used to represent RNA age. This definition describes the temporal properties of RNAs that have undergone diverse life histories and reflects the dynamic state of each ribonucleotide residue, which can be expressed mathematically. Notably, events (including base insertions, base deletions, and base substitutions) are likely to cause RNA to become younger or older when using mean nucleotide ages to represent the RNA age. Although information, including the presence of added markers in RNA, chemical modification structure of the RNA, and the excision of introns in the mRNA in cells, may provide a basis for identifying RNA age, little is known about determining the RNA age of extracellular RNA in the wild. Nonetheless, we believe that RNA age has an important relationship with the diverse biological properties of RNA under intracellular and extracellular conditions. Therefore, our proposed definition of RNA age offers new perspectives for studying dynamic changes in RNA function, RNA aging, ancient RNA, environmental RNA, and the ages of other biomolecules.

Aquatic ecosystems face significant challenges due to increasing human-induced environmental stressors. Recent studies emphasize the role of epigenetic mechanisms in the stress responses and adaptations of organisms to those stressors. Epigenetics influences gene expression, enabling phenotypic plasticity and transgenerational effects. Therefore, understanding the epigenetic responses of aquatic invertebrates to environmental stressors is imperative for aquatic ecosystem research. In this study, we organize the mechanisms of epigenetics in aquatic invertebrates and explore their roles in the responses of aquatic invertebrates to environmental stressors. Furthermore, we discuss the inheritance of epigenetic changes and their influence across generations in aquatic invertebrates. A comprehensive understanding of epigenetic responses is crucial for long-term ecosystem management and conservation strategies in the face of irreversible climate change in aquatic environments. In this review, we synthesize existing knowledge about environmental epigenetics in aquatic invertebrates to provide insights and suggest directions for future research.

The development of chemical scaffolds that target highly conserved MALAT1 RNA received attention due to its significance in splicing, nuclear organization, and gene expression in disease progression pathways. Here, we synthesized a series of N-fused quinazolino-quinazoline-diones via a PIDA-induced C-N coupling methodology to target MALAT1. Interestingly, compound 2z binds to the UUG pocket of a MALAT1 RNA triple-helix through intercalation, evidenced from molecular docking studies, fluorescence-based assay and CD experiments. 2z exhibited cytotoxicity towards MALAT1 overexpressing cancer cells (SKOV-3, IC50 of 8.0 ± 0.4 μM). These findings demonstrated 2z as a MALAT1 RNA triple-helix intercalator with therapeutic potential, offering an important chemical scaffold to understand MALAT1 activity in disease development pathways.

Obviously, the discussion of different factors that could have contributed to the origin of life and evolution is clear speculation, since there is no way of checking the validity of most of the related hypotheses in practice, as the corresponding events not only already happened, but took place in a very distant past. However, there are a few undisputable facts that are present at the moment, such as the existence of a wide variety of living forms and the abundant presence of intrinsically disordered proteins (IDPs) or hybrid proteins containing ordered domains and intrinsically disordered regions (IDRs) in all living forms. Since it seems that the currently existing living forms originated from a common ancestor, their variety is a result of evolution. Therefore, one could ask a logical question of what role(s) the structureless and highly dynamic but vastly abundant and multifunctional IDPs/IDRs might have in evolution. This study represents an attempt to consider various ideas pertaining to the potential roles of protein intrinsic disorder in the origin of life and evolution.

Methanosarcina mazei is a model organism, providing a platform to explore methanoarchaeal regulation mechanisms on the transcriptional and translational level. This study investigates and evaluates various molecular tools to allow inducible gene expression in M. mazei. (i) The TetR/TetO system was utilized to induce expression of a designed antisense RNA directed against sRNA154 allowing to increase transcripts of asRNA154 (500-fold), resulting in a significant decrease of sRNA154 levels (tetracycline-induced knockdown mutant). Strong reduction of sRNA154 was further confirmed in the knockdown mutant by up to 50-fold decreased transcript levels of the genes nifH, glnK1 , and glnA1 , the stability of which is increased by sRNA154. (ii) For translational regulation, an RNA thermometer was designed and first-ever utilized in an archaeon, inserted into the 5'-untranslated region of a reporter gene, which showed enhanced protein expression upon a temperature shift from 30°C to 40°C. (iii) The long 5'-UTR of a trimethylamine (TMA)-inducible polycistronic mRNA was evaluated and studied as a potential genetic tool for induced gene expression on the translational level. However, we discovered TMA-dependent regulation occurs most likely on the transcript level. (iv) A new selection marker (nourseothricin resistance) was established for M. mazei using the streptothricin acetyltransferase gene. Taken together, our findings provide a foundation for future exploration of genetic regulation and inducible gene expression in M. mazei and other methanoarchaea, advancing genetic studies in these organisms and enhancing their potential for biotechnology applications.

Fragile X mental retardation protein (FMRP) is a translational repressor encoded by FMR1. It targets bone morphogenetic protein receptor type II (BMPR2), which regulates granulosa cell (GC) function and follicle development. However, whether this interaction affects folliculogenesis remains unclear. Therefore, this study investigated the potential effect of FMRP-BMPR2 dysregulation in ovarian reserves and infertility. COV434 cells and patient-derived GCs were used to evaluate FMRP and BMPR2 expression. Similarly, FMR1, BMPR2, LIMK1, and SMAD expression were evaluated in GCs with normal (NOR) and poor (POR) ovarian responses. FMRP and BMPR2 were expressed in both cell types. They were co-localized to the nuclear membrane of COV434 cells and cytoplasm of primary GCs. FMR1 silencing increased the mRNA and protein levels of BMPR2. However, the mRNA levels of FMR1 and BMPR2 were significantly lower in the POR group. FMR1 and BMPR2 levels were strongly positively correlated in the NOR group but weakly correlated in the POR group. Additionally, SMAD9 expression was significantly reduced in the POR group. This study highlights the crucial role of FMR1/FMRP in the regulation of BMPR2 expression and its impact on ovarian function. These findings indicate that the disruption of FMRP-BMPR2 interactions may cause poor ovarian responses and infertility.

Ovarian cancer (OC) is a common gynecological disease with a high mortality rate worldwide due to its insidious nature and undetectability at an early stage. The standard treatment, combining platinum‑based chemotherapy with cytoreductive surgery, has suboptimal results. Therefore, early diagnosis of OC is crucial. All cell types secrete extracellular vesicles, particularly exosomes. Exosomes, which contain lipids, proteins, DNA and non‑coding RNAs (ncRNAs), are novel methods of intercellular communication that participate in tumor development and progression. ncRNAs are categorized by size into long ncRNAs (lncRNAs) and small ncRNAs (sncRNAs). sncRNAs further include transfer RNAs, small nucleolar RNAs, PIWI‑interacting RNAs and microRNAs (miRNAs). miRNAs inhibit protein translation and promote messenger RNA (mRNA) cleavage to suppress gene expression. By sponging downstream miRNAs, lncRNAs and circular RNAs can regulate target gene expression, thereby weakening the interactions between miRNAs and mRNAs. Exosomes and exosomal ncRNAs, commonly present in human biological fluids, are promising biomarkers for OC. The present article aimed to review the potential role of exosomal ncRNAs in the diagnosis and prognosis of OC by summarizing the characteristics, processes, roles and isolation methods of exosomes and exosomal ncRNAs.

Long non-coding RNAs (lncRNAs) play crucial roles in the regulation of gene expression and maintenance of genomic integrity through various interactions with DNA, RNA, and proteins. The availability of large-scale sequence data from various high-throughput platforms has opened possibilities to identify, predict, and functionally annotate lncRNAs. As a result, there is a growing demand for an integrative computational framework capable of identifying known lncRNAs, predicting novel lncRNAs, and inferring the downstream regulatory interactions of lncRNAs at the genome-scale. We present ETENLNC (End-To-End-Novel-Long-NonCoding), a user-friendly, integrative, open-source, scalable, and modular computational framework for identifying and analyzing lncRNAs from raw RNA-Seq data. ETENLNC employs six stringent filtration steps to identify novel lncRNAs, performs differential expression analysis of mRNA and lncRNA transcripts, and predicts regulatory interactions between lncRNAs, mRNAs, miRNAs, and proteins. We benchmarked ETENLNC against six existing tools and optimized it for desktop workstations and high-performance computing environments using data from three different species. ETENLNC is freely available on GitHub: https://github.com/EvolOMICS-TU/ETENLNC.

The nucleus is a membrane-bound organelle in eukaryotic cells and plays a crucial role in cellular processes. Visualizing nuclear morphology is essential for investigating nuclear functions and understanding the relationship between nuclear morphological alterations and multiple diseases. Fluorescent dyes have been developed to visualize nuclear morphology, but the selection of red nuclear-labeling fluorescent dyes remains limited (high price, unknown structure, or high toxicity). Herein, we have developed a red ultra-bright nuclear-targeted dye, BPC1, through the engineering of unsymmetrical cyanine dyes derived from D-π-A systems. BPC1 exhibits ultrahigh fluorescence brightness and exceptional cell permeability, and selectively stains nuclear DNA rather than mitochondrial DNA, enabling the visualization of the nucleus in diverse cells at extremely low doses (100 nM) and laser power (0.8 μW). Furthermore, BPC1 is utilized for nuclear staining in blood cells, aiding in the distinct visualization of the white blood cell nucleus and facilitating the identification and enumeration of various leukocyte types. Our study implies considerable commercial potential for BPC1 and underscores its capacity to serve as a powerful tool in life sciences and cell biology research.

Conformational dynamics is crucial for the biological function of RNA molecules and for their potential as therapeutic targets. This meeting report outlines key "take-home" messages that emerged from the presentations and discussions during the CECAM workshop "RNA dynamics from experimental and computational approaches" in Paris, June 26-28, 2023.

The increasing need for mass spectrometric analysis of RNA molecules calls for a better understanding of their gas-phase fragmentation behaviors. In this study, we investigate the effect of terminal phosphate groups on the fragmentation spectra of RNA oligonucleotides (oligos) using high-resolution mass spectrometry (MS). Negative-ion mode collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) were carried out on RNA oligos containing a terminal phosphate group on either end, both ends, or neither end. We find that terminal phosphate groups affect the fragmentation behavior of RNA oligos in a way that is dependent on the precursor charge state and the oligo length. Specifically, for precursor ions of RNA oligos of the same sequence, those with 5'- or 3'-phosphate, or both, have a higher charge state distribution and lose the phosphate group(s) in the form of a neutral (H3PO4 or HPO3) or an anion ([H2PO4]- or [PO3]-) upon CID or HCD. Such a neutral or charged loss is most conspicuous for precursor ions of an intermediate charge state, e.g., 3- for 4-nt oligos or 4- and 5- for 8-nt oligos. This decreases the intensity of sequencing ions (a-, a-B, b-, c-, d-, w-, x-, y-, z-ions) and hence is unfavorable for sequencing by CID or HCD. Removal of terminal phosphate groups by calf intestinal alkaline phosphatase improved MS analysis of RNA oligos. Additionally, the intensity of a fragment ion at m/z 158.925, which we identified as a dehydrated pyrophosphate anion ([HP2O6]-), is markedly increased by the presence of a terminal phosphate group. These findings expand the knowledge base necessary for software development for MS analysis of RNA.

RNA molecules play a crucial role in various biological processes, with their functionality closely tied to their structures. The remarkable advancements in machine learning techniques for protein structure prediction have shown promise in the field of RNA structure prediction. In this perspective, we discuss the advances and challenges encountered in constructing machine learning-based models for RNA structure prediction. We explore topics including model building strategies, specific challenges involved in predicting RNA secondary (2D) and tertiary (3D) structures, and approaches to these challenges. In addition, we highlight the advantages and challenges of constructing RNA language models. Given the rapid advances of machine learning techniques, we anticipate that machine learning-based models will serve as important tools for predicting RNA structures, thereby enriching our understanding of RNA structures and their corresponding functions.

Dysregulation of the epigenomic landscape of tumor cells has been implicated in the pathogenesis of pancreatic cancer. However, these alterations are not only restricted to neoplastic cells. The behavior of other cell populations in the tumor stroma such as cancer-associated fibroblasts, immune cells, and others are mostly regulated by epigenetic pathways. Here, we present an overview of the main cellular and acellular components of the pancreatic cancer tumor microenvironment and discuss how the epigenetic mechanisms operate at different levels in the stroma to establish a differential gene expression to regulate distinct cellular phenotypes contributing to pancreatic tumorigenesis.

MicroRNAs are small non-coding RNAs evolutionary conserved molecules. They regulate cellular processes, including RNA silencing, post-translational gene expression and neurodegeneration. MicroRNAs are involved with human diseases such as cancer, Alzheimer's disease (AD) and others. Interestingly, cerebrospinal fluids (CSF) and the blood of AD patients have altered expressions of many RNAs, which may serve as potential peripheral biomarkers. The intensive investigation from our lab revealed that microRNA-455-3 P (miR-455-3p) is a strong candidate as a potential biomarker and therapeutic target for AD. Several genes implicated in the pathogenesis of AD are directly targeted by miR-455-3p. Several years of our lab research revealed that miR-455-3p regulates important physiological processes associated with AD, such as the processing of the amyloid precursor protein (APP), TGF-β signaling, the regulation of oxidative stress, mitochondrial biogenesis, and synaptic damages. The expression of miR-455-3p in mild cognitive impaired subjects and AD patients pointed out its involvement in AD progression. Recently, our lab generated both transgenic and knockout mice for miR-455-3p. Interestingly miR-455-3p transgenic mice showed superior cognitive learning, improved memory and extended lifespan compared to age matched wild-type mice, whereas miR-455-3-p knockout mice showed cognitive decline and reduced lifespan. Information derived from mouse models further demonstrated the advantageous impact of miR-455-3p on dendritic growth, synaptogenesis, and mitochondrial biogenesis in preventing the onset and progression of AD. The identification of miR-455-3p as a biomarker was suggested by its presence in postmortem AD brains, B-lymphocytes, and fibroblasts. Our hypothesis that miR-455-3p could be a peripheral biomarker and therapeutic target for AD.

The role of ribonucleic acid (RNA) in biology continues to grow, but insight into important aspects of RNA behavior is lacking, such as dynamic structural ensembles in different environments, how flexibility is coupled to function, and how function might be modulated by small molecule binding. In the case of proteins, much progress in these areas has been made by complementing experiments with atomistic simulations, but RNA simulation methods and force fields are less mature. It remains challenging to generate stable RNA simulations, even for small systems where well-defined, thermostable structures have been established by experiments. Many different aspects of RNA energetics have been adjusted in force fields, seeking improvements that are transferable across a variety of RNA structural motifs. In this work, the role of weak CH···O interactions is explored, which are ubiquitous in RNA structure but have received less attention in RNA force field development. By comparing data extracted from high-resolution RNA crystal structures to energy profiles from quantum mechanics and force field calculations, it is shown that CH···O interactions are overly repulsive in the widely used Amber RNA force fields. A simple, targeted adjustment of CH···O repulsion that leaves the remainder of the force field unchanged was developed. Then, the standard and modified force fields were tested using molecular dynamics (MD) simulations with explicit water and salt, amassing over 300 μs of data for multiple RNA systems containing important features such as the presence of loops, base stacking interactions as well as canonical and noncanonical base pairing. In this work and others, standard force fields lead to reproducible unfolding of the NMR-based structures. Including a targeted CH···O adjustment in an otherwise identical protocol dramatically improves the outcome, leading to stable simulations for all RNA systems tested.

Accurately predicting patient outcomes is essential for optimizing treatment and improving outcomes in pediatric acute myeloid leukemia (AML). In recent years, microRNAs have emerged as a promising prognostic marker, with a growing body of evidence supporting their potential predictive value. We systematically reviewed all previous studies that have analyzed the expression of microRNAs as predictors of survival in pediatric AML and found 16 microRNAs and 4 microRNA signatures previously proposed as predictors of survival. We then used a public access cohort of 1414 pediatric AML patients from the TARGET project to develop a new predictive model using penalized lasso Cox regression based on microRNA expression. Here we propose a new score based on a 37-microRNA signature that is associated with AML and is able to predict survival more accurately than previous microRNA-based methods.

RNA constitutes a large fraction of chromatin. Spatial distribution and functional relevance of most of RNA-chromatin interactions remain unknown. We established a landscape analysis of RNA-chromatin interactions in human acute myeloid leukemia (AML). In total more than 50 million interactions were captured in an AML cell line. Protein-coding mRNAs and long non-coding RNAs exhibited a substantial number of interactions with chromatin in cis suggesting transcriptional activity. In contrast, small nucleolar RNAs (snoRNAs) and small nuclear RNAs (snRNAs) associated with chromatin predominantly in trans suggesting chromatin specific functions. Of note, snoRNA-chromatin interaction was associated with chromatin modifications and occurred independently of the classical snoRNA-RNP complex. Two C/D box snoRNAs, namely SNORD118 and SNORD3A, displayed high frequency of trans-association with chromatin. The transcription of SNORD118 and SNORD3A was increased upon leukemia transformation and enriched in leukemia stem cells, but decreased during myeloid differentiation. Suppression of SNORD118 and SNORD3A impaired leukemia cell proliferation and colony forming capacity in AML cell lines and primary patient samples. Notably, this effect was leukemia specific with less impact on healthy CD34+ hematopoietic stem and progenitor cells. These findings highlight the functional importance of chromatin-associated RNAs overall and in particular of SNORD118 and SNORD3A in maintaining leukemia propagation.

Extracellular vesicles (EVs), which include small EVs such as exosomes, play a critical role in intercellular communication and are produced by both cancer and non-cancer cells. Several studies have shown that cancer cells exploit various strategies to regulate the biogenesis, composition, and functions of EVs primarily to promote cancer progression. Given that exosomes originate from major sorting hubs at the limiting membrane of endosomes, they are central to a signaling network that connects external stimuli with intrinsic tumor cell features. Exosomes contain diverse repertoires of molecular cargos, such as proteins, lipids, and nucleic acids, which determine their heterogeneity and functional properties in cancer progression. Therefore, targeting exosome biogenesis will enhance our understanding of tumorigenesis and also promote the discovery of novel approaches for cancer therapy. In this chapter we summarize the machinery of exosome biogenesis and the local, distant, and systemic effects of exosomes released by cancer cells. Furthermore, we explore how these exosomes regulate the anti-tumor immune response and epigenetic mechanisms to sustain cancer progression and their implications in cancer prevention and treatment.

Lung cancer patients treated with targeted therapies frequently respond well but invariably relapse due to the development of drug resistance. Drug resistance is in part mediated by a subset of cancer cells termed "drug-tolerant persisters" (DTPs), which enter a dormant, slow-cycling state that enables them to survive drug exposure. DTPs also exhibit stem cell-like characteristics, broad epigenetic reprogramming, altered metabolism, and a mutagenic phenotype mediated by adaptive mutability. While several studies have characterised the transcriptional changes that lead to the altered phenotypes exhibited in DTPs, these studies have focused predominantly on protein coding changes. As long non-coding RNAs (lncRNAs) are also implicated in the phenotypes altered in DTPs, it is likely that they play a role in the biology of drug tolerance. In this review, we outline how lncRNAs may contribute to the key characteristics of DTPs, their potential roles in tolerance to targeted therapies, and the emergence of genetic resistance in lung adenocarcinoma.

Candida albicans is known for its pathogenicity, although it lives within the human body as a commensal member. The commensal nature of C. albicans is well controlled and regulated by the host's immune system as they live in the harmonized microenvironment. However, the development of certain unusual microhabitat conditions (change in pH, co-inhabiting microorganisms' population ratio, debilitated host-immune system) pokes this commensal fungus to transform into a pathogen in such a way that it starts to propagate very rapidly and tries to breach the epithelial barrier to enter the host's systemic circulations. In addition, Candida is infamous as a major nosocomial (hospital-acquired infection) agent because it enters the human body through venous catheters or medical prostheses. The hysterical mode of C. albicans growth builds its microcolony or biofilm, which is pathogenic for the host. Biofilms propose additional resistance mechanisms from host immunity or extracellular chemicals to aid their survival. Differential gene expressions and regulations within the biofilms cause altered morphology and metabolism. The genes associated with adhesiveness, hyphal/pseudo-hyphal growth, persister cell transformation, and biofilm formation by C. albicans are controlled by myriads of cell-signaling regulators. These genes' transcription is controlled by different molecular determinants like transcription factors and regulators. Therefore, this review has focused discussion on host-immune-sensing molecular determinants of Candida during biofilm formation, regulatory descriptors (secondary messengers, regulatory RNAs, transcription factors) of Candida involved in biofilm formation that could enable small-molecule drug discovery against these molecular determinants, and lead to disrupt the well-structured Candida biofilms effectively.

Rapid and sensitive RNA detection is of great value in diverse areas, ranging from biomedical research to clinical diagnostics. Existing methods for RNA detection often rely on reverse transcription (RT) and DNA amplification or involve a time-consuming procedure and poor sensitivity. Herein, we proposed a CRISPR/Cas12a-enabled amplification-free assay for rapid, specific, and sensitive RNA diagnostics. This assay, which we termed T7/G4-CRISPR, involved the use of a T7-powered nucleic acid circuit to convert a single RNA target into numerous DNA activators via toehold-mediated strand displacement reaction and T7 exonuclease-mediated target recycling amplification, followed by activating Cas12a trans-cleavage of the linker strands inhibiting split G-Quadruplex (G4) assembly, thereby inducing fluorescence attenuation proportion to the input RNA target. We first performed step-by-step validation of the entire assay process and optimized the reaction parameters. Using the optimal conditions, T7/G4-CRISPR was capable of detecting as low as 3.6 pM target RNA, obtaining ∼100-fold improvement in sensitivity compared with the most direct Cas12a assays. Meanwhile, its excellent specificity could discriminate single nucleotide variants adjacent to the toehold region and allow species-specific pathogen identification. Furthermore, we applied it for analyzing bacterial 16S rRNA in 40 clinical urine samples, exhibiting a sensitivity of 90% and a specificity of 100% when validated by RT-quantitative PCR. Therefore, we envision that T7/G4-CRISPR will serve as a promising RNA sensing approach to expand the toolbox of CRISPR-based diagnostics.