RNA molecules display distinctive organization at nuclear speckles.

RNA molecules often play critical roles in assisting the formation of membraneless organelles in eukaryotic cells. Yet, little is known about the organization of RNAs within membraneless organelles. Here, using super-resolution imaging and nuclear speckles as a model system, we demonstrate that different sequence domains of RNA transcripts exhibit differential spatial distributions within speckles. Specifically, we image transcripts containing a region enriched in binding motifs of serine/arginine-rich (SR) proteins and another region enriched in binding motifs of heterogeneous nuclear ribonucleoproteins (hnRNPs). We show that these transcripts localize to the outer shell of speckles, with the SR motif-rich region localizing closer to the speckle center relative to the hnRNP motif-rich region. Further, we identify that this intra-speckle RNA organization is driven by the strength of RNA-protein interactions inside and outside speckles. Our results hint at novel functional roles of nuclear speckles and likely other membraneless organelles in organizing RNA substrates for biochemical reactions.

Transcriptome-wide mRNA condensation precedes stress granule formation and excludes stress-induced transcripts.

Stress-induced condensation of mRNA and proteins into stress granules is conserved across eukaryotes, yet the function, formation mechanisms, and relation to well-studied conserved transcriptional responses remain largely unresolved. Stress-induced exposure of ribosome-free mRNA following translational shutoff is thought to cause condensation by allowing new multivalent RNA-dependent interactions, with RNA length and associated interaction capacity driving increased condensation. Here we show that, in striking contrast, virtually all mRNA species condense in response to multiple unrelated stresses in budding yeast, length plays a minor role, and instead, stress-induced transcripts are preferentially excluded from condensates, enabling their selective translation. Using both endogenous genes and reporter constructs, we show that translation initiation blockade, rather than resulting ribosome-free RNA, causes condensation. These translation initiation-inhibited condensates (TIICs) are biochemically detectable even when stress granules, defined as microscopically visible foci, are absent or blocked. TIICs occur in unstressed yeast cells, and, during stress, grow before the appearance of visible stress granules. Stress-induced transcripts are excluded from TIICs primarily due to the timing of their expression, rather than their sequence features. Together, our results reveal a simple system by which cells redirect translational activity to newly synthesized transcripts during stress, with broad implications for cellular regulation in changing conditions.

Indole-based NNN donor Schiff base ligand and its complexes: Sonication-assisted synthesis, characterization, DNA binding, anti-cancer evaluation and in-vitro biological assay.

A novel indole based NNN donor Schiff base ligand and its Ni(II), Zn(II) and Cd(II) complexes have been synthesized using sonication-assisted method which is a highly efficient eco-friendly mechanism. The synthesized complexes have been characterized using elemental analysis, UV-Vis spectroscopy, mass spectrometry, FT-IR, and NMR and are optimized using DFT approach, which provided their theoretical framework. The stoichiometry between the ligand and the metal ions was also determined using Job's method. The thermogravimetric (TGA/DSC) analyses confirm the stability for all complexes at room temperature followed by thermal decomposition in different steps. DNA binding activities have been assessed by employing UV-visible and fluorescence spectra using the CT-DNA. The estimated intrinsic binding constant (Kb) for NiL, ZnL, and CdL complexes was 6.00 × 105, 5.58 × 105, and 4.7 × 105, respectively. In accordance with the Kb value, the quenching constant (Ksv) values of NiL, ZnL, and CdL are 5.59 × 105 M-1, 4.3 × 105 M-1, and 4.08 × 105 M-1 respectively. The anticancer properties have been assessed using MTT Assay. It has been found that the Ni(II) complex (NiL) is the most potent among the series with IC50 of 169 µg/mL. An in-vitro antioxidant experiment using DPPH was used to evaluate the synthesizedcomplexes' ability to scavenge free radicals. The findings indicated that the complexes exhibited notable antioxidant properties. The antioxidant property ZnL has been found to be the highest with an IC50 of 2.91 µg/mL and it follows the order is ZnL > NiL > CdL > L. Using the egg albumin denaturation technique, the anti-inflammatory property have been assessed, and the amount of protein denaturation inhibition has been computed. NiL has the highest % inhibition among the series studied. Comparatively, the metal complexes have been reported to exhibit higher biological activities than the prepared Schiff base ligand. The reason for the excellent biological properties observed in the metal complexes could be attributed to the incorporation of the electron-withdrawing CH3COO- during complexation. Molecular docking studies have been performed on the 2GYT protein and it has been found that the complexes have excellent binding affinity, with NiL having the lowest binding energy of -6.93 Kcal mol-1. The values suggested that NiL is more effective against HePG2 cancer cells, which is also in accordance with the MTT Assay results.

Dynamics of RNA localization to nuclear speckles are connected to splicing efficiency.

Nuclear speckles, a type of membraneless nuclear organelle in higher eukaryotic cells, play a vital role in gene expression regulation. Using the reverse transcription-based RNA-binding protein binding sites sequencing (ARTR-seq) method, we study human transcripts associated with nuclear speckles. We identify three gene groups whose transcripts demonstrate different speckle localization properties and dynamics: stably enriched in nuclear speckles, transiently enriched in speckles at the pre-mRNA stage, and not enriched in speckles. Specifically, we find that stably-enriched transcripts contain inefficiently spliced introns. We show that nuclear speckles specifically facilitate splicing of speckle-enriched transcripts. We further reveal RNA sequence features contributing to transcript speckle localization, underscoring a tight interplay between genome organization, RNA cis-elements, and transcript speckle enrichment, and connecting transcript speckle localization with splicing efficiency. Finally, we show that speckles can act as hubs for the regulated retention of introns during cellular stress. Collectively, our data highlight a role of nuclear speckles in both co- and post-transcriptional splicing regulation.

FishSegSSL: A Semi-Supervised Semantic Segmentation Framework for Fish-Eye Images.

The application of large field-of-view (FoV) cameras equipped with fish-eye lenses brings notable advantages to various real-world computer vision applications, including autonomous driving. While deep learning has proven successful in conventional computer vision applications using regular perspective images, its potential in fish-eye camera contexts remains largely unexplored due to limited datasets for fully supervised learning. Semi-supervised learning comes as a potential solution to manage this challenge. In this study, we explore and benchmark two popular semi-supervised methods from the perspective image domain for fish-eye image segmentation. We further introduce FishSegSSL, a novel fish-eye image segmentation framework featuring three semi-supervised components: pseudo-label filtering, dynamic confidence thresholding, and robust strong augmentation. Evaluation on the WoodScape dataset, collected from vehicle-mounted fish-eye cameras, demonstrates that our proposed method enhances the model's performance by up to 10.49% over fully supervised methods using the same amount of labeled data. Our method also improves the existing image segmentation methods by 2.34%. To the best of our knowledge, this is the first work on semi-supervised semantic segmentation on fish-eye images. Additionally, we conduct a comprehensive ablation study and sensitivity analysis to showcase the efficacy of each proposed method in this research.

Structural Stability and Conformational Dynamics of Cytochrome c in Hydrated Deep Eutectic Solvents.

Many deep eutectic solvents (DESs) are currently being explored as environment-friendly media for biorelated applications. As an understanding of the effect of these solvents on the structure of biomolecules is crucial for these applications, we study how two DESs comprising trimethylglycine (TMG) and ethylene glycol (EG) or glycerol (GL) influence the structural stability and conformational dynamics of cytochrome c (Cytc) using single-molecule-based fluorescence correlation spectroscopy (FCS) technique and several other ensemble-based biophysical methods. The FCS studies on A488-labeled Cytc enable an estimation of the size (20.5 ± 1.5 Å) of the protein and capture its conformational dynamics (54 ± 2 µs) in aqueous buffered solution. It is observed that both size and conformational dynamics of the protein are influenced in the presence of the DESs, but this effect is more pronounced in the case of TMG-EG. The ensemble measurements on both labeled and wild-type Cytc reveal that the protein structure is unfolded completely by TMG-EG, whereas the structure is slightly altered by TMG-GL. The results suggest that the behavior of Cytc in hydrated DESs is determined by the strength of interactions between the DES constituents as well as that between the constituents and the water molecules present in the system.

Complete Solvation Dynamics of Coumarin 153 in Tetraalkylammonium Bromide-Based Deep Eutectic Solvents.

Deep eutectic solvents (DESs) are novel environment-friendly media for a variety of applications. In order to obtain insight into the structure and dynamics of some less-explored DESs comprising ethylene glycol and tetraalkylammonium bromide salts with variable alkyl chain length, we have captured complete dynamics occurring in these solvents in a timescale of few femtoseconds to several nanoseconds by monitoring the time-dependent fluorescence Stokes shift of coumarin 153 employing a combination of time-correlated single-photon counting and fluorescence upconversion techniques. The solvent response function constructed from the measured data reveals a sub-picosecond component (∼0.8 ps, 20-35%) in addition to a slow component (180-475 ps) with a distribution of relaxation time. The slow time component is found to be strongly dependent on the viscosity of the medium, indicating that it arises from the diffusive motions of the solvent constituents into and out of the solvation shell, whereas the ultrafast time component, which is nearly independent of the solvent viscosity, arises from fast local motions of the constituents in the immediate vicinity of the solute molecule.

Insights into the Folding Pathway of a c-MYC-Promoter-Based i-Motif DNA in Crowded Environments at the Single-Molecule Level.

Cytosine-rich DNA sequences fold into secondary structures called i-Motifs, which are usually stable at acidic pH. However, molecular crowding agents, such as poly(ethylene glycol) (PEG), are known to facilitate the formation of these structures even at neutral pH. As crowding mimics the intracellular environment and not much is known about the folding pathway of i-Motifs in such constrained media, we have probed, in detail, the conformational changes of a 22-mer c-MYC-promoter-based C-rich sequence (Py22) in the presence of PEG, employing Förster resonance energy transfer and fluorescence lifetime measurements at the single-molecule level. We find that the folding process is not a simple two-state transition between a random coil and a folded i-Motif structure. Rather, it involves a partially folded conformation as an intermediate in which the bases are not as efficiently stacked as in the completely folded i-Motif form. The relative population of each species is governed by the size and concentration of PEG, and 30% (w/w) PEG6000 is the optimum condition for the folding of Py22. Under this condition, ∼80% of Py22 exists in the fully folded i-Motif form and ∼20% of it is in the partially folded state.

Liquid Structure and Dynamics of Tetraalkylammonium Bromide-Based Deep Eutectic Solvents: Effect of Cation Chain Length.

Deep eutectic solvents (DESs) have emerged in recent years as environmentally sustainable media across several fields. However, knowledge of liquid structure, dynamics, and solute-solvent interactions in many DESs that is essential for exploiting their potential is still lacking. In this work, we make an attempt to obtain some insight into these aspects of a set of less-explored DESs comprising tetraalkylammonium bromide salts and ethylene glycol (EG) by monitoring the fluorescence response of some carefully chosen dipolar (C153 and 4-AP) and nonpolar (9-PA) solutes in these media. Specifically, we have studied the translational and rotational diffusion dynamics of these molecular systems using single-molecule-based fluorescence correlation spectroscopy technique and ensemble-based time-resolved fluorescence anisotropy measurements. These results point to spatial and dynamic heterogeneity of these DESs, which becomes prominent in systems comprising cations with a longer alkyl chain length. This study reveals that diffusion dynamics of the probe molecules is determined not only by the solvent bulk viscosity but also dependent on their microenvironments and solute-solvent interactions experienced in these media.

Interactions between a Bioflavonoid and c-MYC Promoter G-Quadruplex DNA: Ensemble and Single-Molecule Investigations.

Small molecules capable of stabilizing the G-quadruplex structure of the nuclease hypersensitivity element III1 (NHE III1) are useful in controlling the overexpression of the c-MYC oncogene. In this study, we have probed the interactions of a 22-mer c-MYC promoter quadruplex-forming sequence (Pu22) with a bioflavonoid 3,4',5,7-tetrahydroxyflavone, commonly known as kaempferol (KF). Ensemble fluorescence resonance energy transfer experiments on labeled Pu22 indicate that KF decreases the affinity of the former toward its complimentary strand, suggesting the stabilization of the quadruplex structure of Pu22. Considering that binding dynamics plays an important role in supramolecular interactions, there is hardly any information on this aspect for quadruplex-flavonoid systems; we have studied the kinetics of KF-Pu22 complexation and decomplexation processes on the single-molecule level by employing fluorescence correlation spectroscopy technique. The binding dynamics is characterized by a fast relaxation time of 10-50 µs. This leads to a high association rate constant ( k+) of ∼109 M-1 s-1, which is close to the pure diffusion controlled limit. However, it is the low dissociation rate constant ( k-) of ∼104 s-1 that is mainly responsible for the stability of the KF-Pu22 complex. Molecular docking study shows that KF binds near the 3'-end of Pu22 by forming several H-bonds with the bases. These findings suggest that KF is a potential binder of the c-MYC promoter quadruplex DNA and can be useful in anticancer therapies.

Ground- and Excited-State Interactions of a Psoralen Derivative with Human Telomeric G-Quadruplex DNA.

G-quadruplex DNA has been a recent target for anticancer agents, and its binding interactions with small molecules, often used as anticancer drugs, have become an important area of research. Considering that psoralens have long been studied in the context of duplex DNA but that very little is known about their potential as G-quadruplex binders and their excited-state interaction with the latter has not been explored, we have studied herein the binding of a planar water-soluble psoralen derivative, 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), with the 22-mer human telomeric G-quadruplex-forming sequence, AGGG(TTAGGG)3, labeled here as (hTel22), and investigated the consequences of photoexcitation of AMT by calorimetric and spectroscopic techniques. The results show an enthalpy-driven 1:1 binding of AMT with hTel22 via end-stacking mode. Fluorescence quenching experiments on 6-fluorescein amidite-labeled oligomers indicate that the binding site is nearer to the 3' end of hTel22 in the diagonal loop region. Femtosecond time-resolved transient absorption measurements indicate electron transfer from the guanine moiety of hTel22 to photoexcited AMT, leading to the formation of a radical pair species (AMT•-G•+), which survives for 30 ps and is favored by a parallel/quasi-parallel orientation between the two. The findings reveal psoralens as a prospective class of compounds for the development of anticancer therapeutics by targeting the G-quadruplex DNA.

Influence of Divalent Counterions on the Dynamics in DNA as Probed by Using a Minor-Groove Binder.

DNA dynamics, to which water, counterions, and DNA motions contribute, is a topic of considerable interest because it is closely related to the efficiency of biological functions performed by it. Simulation studies and experiments suggest that the counterion dynamics in DNA probed by a minor-groove binder are similar for various monovalent counterions. To date, the influence on DNA dynamics of higher-valence counterions, which are also present around DNA and are known to bind more strongly to it than monovalent ions, has not been studied. Herein we investigated DNA dynamics in the presence of Mg2+ and Ca2+ , chosen for their relative abundance in cells, by using minor-groove binder 4',6-diamidino-2-phenylindole (DAPI) as a fluorescence probe. The dynamics, as measured from the time-resolved fluorescence Stokes shifts of DAPI bound to calf thymus DNA on a subpicosecond-to-nanosecond timescale, were found to be very similar in the presence of both the divalent ions and Na+ ions. The observation is explained by considering the screening of the electric field of the divalent ion by its hydration shell, preferential binding of the ions to the phosphate groups, and displacement of ions from the minor groove by DAPI due to the stronger binding interaction of the latter. Furthermore, the similarity of our results in the presence of Na+ to those reported for smaller oligonucleotides suggests that the chain length of DNA does not influence the DNA dynamics.

Photoinduced 2-way electron transfer in composites of metal nanoclusters and semiconductor quantum dots.

In order to explore the potential of nanocomposites comprising semiconductor quantum dots (QDs) and metal nanoclusters (NCs) in photovoltaic and catalytic applications, the interaction between CdTe QDs and gold NCs, Au10 and Au25, stabilized by histidine, bovine serum albumin (BSA) and glutathione, is studied by an ultrafast transient absorption (TA) technique. Temporal and spectral studies of the transients reveal photoinduced 2-way electron transfer between the two constituents of the nanocomposites, where Au NCs, which generally act as electron donors when used as photosensitizers, perform the role of the efficient electron acceptor. Interestingly, it is found that the electron transfer dynamics in these composites is governed not by the distance of separation of the constituents but by the nature of the surface capping ligands. Despite a large separation between the QDs and NCs in a giant BSA-capped system, a higher electron transfer rate in this composite suggests that unlike other smaller capping agents, which act more like insulators, BSA allows much better electron conduction, as indicated previously.

Contrasting Response of Two Dipolar Fluorescence Probes in a Leucine-Based Organogel and Its Implications.

The microenvironments of a leucine-based organogel are probed by monitoring the fluorescence behavior of coumarin 153 (C153) and 4-aminophthalimide (AP). The steady-state data reveals distinctly different locations of the two molecules in the gel. Whereas AP resides close to the hydroxyl moieties of the gelator and engages in hydrogen-bonding interactions, C153 is found in bulk-toluene-like regions. In contrast to C153, AP exhibits excitation-wavelength-dependent emission, indicating that the environments of the hydrogen-bonded AP molecules are not all identical. A two-component fluorescence decay of AP in gel, unlike C153, supports this model. A time-resolved fluorescence anisotropy study of the rotational motion of the molecules also reveals the strong association of only AP with the gelator. That AP influences the critical gelation concentration implies its direct involvement in the gel-formation process. The results highlight the importance of guest-gelator interactions in gels containing guest molecules.

Overview of computational vaccinology: vaccine development through information technology.

Pathogenic organisms, causes of various infectious diseases, possess a rich repository of antigenic proteins that engender an immune response in a host. These types of diseases are usually treated with the use of pharmaceuticals; unfortunately, many of these also have a potential to induce fatal side effects, especially allergic responses in the diseased host. In addition, many pathogens evolve (by selective survival) single or multi-drug resistance (MDR). Therefore, a means to prevent the host from becoming susceptible to the pathogen from the onset, rather than trying to devise pharmacologic protocols to treat an ongoing infection, are increasingly seen as desirable to reduce the incidence of infectious diseases altogether. To this end, cost-effective development and use of "safe" vaccines is key. This paper provides an overview on the new and expanding area of computational vaccinology and a brief background on pathogen antigenicity, identification of pathogen-specific antigens, and screening of candidate antigens using various tools and databases developed in the recent past.