Multicenter integrated analysis of noncoding CRISPRi screens.

The ENCODE Consortium's efforts to annotate noncoding cis-regulatory elements (CREs) have advanced our understanding of gene regulatory landscapes. Pooled, noncoding CRISPR screens offer a systematic approach to investigate cis-regulatory mechanisms. The ENCODE4 Functional Characterization Centers conducted 108 screens in human cell lines, comprising >540,000 perturbations across 24.85 megabases of the genome. Using 332 functionally confirmed CRE-gene links in K562 cells, we established guidelines for screening endogenous noncoding elements with CRISPR interference (CRISPRi), including accurate detection of CREs that exhibit variable, often low, transcriptional effects. Benchmarking five screen analysis tools, we find that CASA produces the most conservative CRE calls and is robust to artifacts of low-specificity single guide RNAs. We uncover a subtle DNA strand bias for CRISPRi in transcribed regions with implications for screen design and analysis. Together, we provide an accessible data resource, predesigned single guide RNAs for targeting 3,275,697 ENCODE SCREEN candidate CREs with CRISPRi and screening guidelines to accelerate functional characterization of the noncoding genome.

Freestanding, Freeform Metamolecule Fibers Tailoring Artificial Optical Magnetism.

Metamolecule clusters support various unique types of artificial electromagnetism at optical frequencies. However, the technological challenges regarding the freeform fabrication of freestanding metamolecule clusters with programmed geometries and multiple compositions remain unresolved. Here, the freeform, freestanding raspberry-like metamolecule (RMM) fibers based on the directional guidance of a femtoliter meniscus are presented, resulting in the evaporative co-assembly of silica nanoparticles and gold nanoparticles with the aid of 3D nanoprinting. This method offers a facile and universal pathway to shape RMM fibers in 3D, enabling versatile manipulation of near- and far-field characteristics. In particular, the authors demonstrate the ability to decrease the scattering of the millimeter-scale RMM fiber in visible spectrum. In addition, the influence of electric and magnetic dipole modes on the directional scattering of RMM fibers is investigated. These experiments show that the magnetic response of an individual RMM can be controlled by adjusting the filling factor of gold nanoparticles. The authors anticipate that this method will allow for unrestricted design and realization of nanophotonic structures, surpassing the limitations of conventional fabrication processes.

OGD/R-induced ferroptosis and pyroptosis in retinal pigment epithelium cells: Role of PLD1 and PLD2 modulation.

This study investigated the role of phospholipase D (PLD) in retinal ischemia-reperfusion (I/R) injury using an oxygen-glucose deprivation/reperfusion (OGD/R) model commonly used in retinal I/R injury research. To create an in vitro cellular I/R model, pharmacological inhibitors and small interfering RNA (siRNA) were used to target PLD1 and PLD2 in retinal pigment epithelial (RPE) cells. Treatment with PLD inhibitors and siRNA reduced reactive oxygen species (ROS) and malondialdehyde (MDA) induced by OGD/R in RPE cells and increased the levels of superoxide dismutase (SOD) and glutathione (GSH), indicating a reduction in oxidative damage and improvement in the antioxidant system. Next, we showed that inhibiting PLD1 or PLD2 reduced intracellular iron levels and lipid peroxidation, which are critical factors in ferroptosis. Additionally, PLD1 and PLD2 modulated the expression of proteins involved in the regulation of ferroptosis, including GPX4, SLC7A11, FTH1, and ACSL4. We also investigated the roles of PLD1 and PLD2 in preventing pyroptosis, another form of programmed cell death associated with inflammation. Our study found that OGD/R significantly increased the production of pro-inflammatory cytokines and activated caspase-1, NLRP3, ASC, cleaved-caspase 1 (C-caspase-1), and GSDMD-N in RPE cells, indicating pyroptosis induction. However, PLD1 and PLD2 inhibition or knockdown significantly inhibited the production of pro-inflammatory cytokines and activation of the NLRP3 inflammasome, Taken together, our findings support the hypothesis that the PLD signaling pathway plays a key role in OGD/R-induced ferroptosis and pyroptosis induction and may be a potential therapeutic target for preventing or treating retinal dysfunction and degeneration.

Induction of Browning in White Adipocytes: Fucoidan Characterization and Gold Nanoparticle Synthesis from Undaria pinnatifida Sporophyll Extract.

Seaweed extracts and their specific polysaccharides are widely known for their ability to act as reducing and capping agents during nanoparticle synthesis. Their application is highly favored in green synthesis methods, owing to their eco-friendliness, cost-effectiveness, and remarkable time and energy efficiency. In this study, fucoidan extracted from Undaria pinnatifida sporophyll (UPS) is introduced as a polysaccharide that effectively serves as a dual-function reducing and capping agent for the synthesis of gold nanoparticles (AuNPs). Results from various analyses indicate that AuNPs derived from UPS extract display a uniform spherical shape with an average size of 28.34 ± 1.15 nm and a zeta potential of -37.49 ± 2.13 mV, conclusively confirming the presence of Au. The FT-IR spectra distinctly revealed the characteristic fucoidan bands on the stabilized UPS-AuNPs surface. A 1H-NMR analysis provided additional confirmation by revealing the presence of specific fucoidan protons on the UPS-AuNPs surface. To comprehensively evaluate the impact of UPS extract, UPS-AuNPs, and fucoidan on the biological properties of adipocytes, a rigorous comparative analysis of lipid droplet formation and morphology was conducted. Our findings revealed that adipocytes treated with UPS extract, fucoidan, and UPS-AuNPs, in that order, exhibited a reduction in the total lipid droplet surface area, maximum Ferret diameter, and overall Nile red staining intensity when compared to mature white adipocytes. Furthermore, our analysis of the effects of UPS extracts, UPS-AuNPs, and fucoidan on the expression of key markers associated with white adipose tissue browning, such as UCP1, PGC1a, and PRDM16, demonstrated increased mRNA and protein expression levels in the following order: UPS-AuNPs > fucoidan > UPS extracts. Notably, the production of active mitochondria, which play a crucial role in enhancing energy expenditure in beige adipocytes, also increased in the following order: UPS-AuNPs > fucoidan > UPS extract. These findings underscore the pivotal role of UPS extract, fucoidan, and UPS-AuNPs in promoting adipocyte browning and subsequently enhancing energy expenditure.

Understanding the Role of M13 Bacteriophage Thin Films on a Metallic Nanostructure through a Standard and Dynamic Model.

The dynamic and surface manipulation of the M13 bacteriophage via the meeting application demands the creation of a pathway to design efficient applications with high selectivity and responsivity rates. Here, we report the role of the M13 bacteriophage thin film layer that is deposited on an optical nanostructure involving gold nanoparticles/SiO2/Si, as well as its influence on optical and geometrical properties. The thickness of the M13 bacteriophage layer was controlled by varying either the concentration or humidity exposure levels, and optical studies were conducted. We designed a standard and dynamic model based upon three-dimensional finite-difference time-domain (3D FDTD) simulations that distinguished the respective necessity of each model under variable conditions. As seen in the experiments, the origin of respective peak wavelength positions was addressed in detail with the help of simulations. The importance of the dynamic model was noted when humidity-based experiments were conducted. Upon introducing varied humidity levels, the dynamic model predicted changes in plasmonic properties as a function of changes in NP positioning, gap size, and effective index (this approach agreed with the experiments and simulated results). We believe that this work will provide fundamental insight into understanding and interpreting the geometrical and optical properties of the nanostructures that involve the M13 bacteriophage. By combining such significant plasmonic properties with the numerous benefits of M13 bacteriophage (like low-cost fabrication, multi-wavelength optical characteristics devised from a single structure, reproducibility, reversible characteristics, and surface modification to suit application requirements), it is possible to develop highly efficient integrated plasmonic biomaterial-based sensor nanostructures.

Three-Dimensional Plasmonic Nanocluster-Driven Light-Matter Interaction for Photoluminescence Enhancement and Picomolar-Level Biosensing.

Plasmonic nanoparticle clusters promise to support unique engineered electromagnetic responses at optical frequencies, realizing a new concept of devices for nanophotonic applications. However, the technological challenges associated with the fabrication of three-dimensional nanoparticle clusters with programmed compositions remain unresolved. Here, we present a novel strategy for realizing heterogeneous structures that enable efficient near-field coupling between the plasmonic modes of gold nanoparticles and various other nanomaterials via a simple three-dimensional coassembly process. Quantum dots embedded in the plasmonic structures display ∼56 meV of a blue shift in the emission spectrum. The decay enhancement factor increases as the total contribution of radiative and nonradiative plasmonic modes increases. Furthermore, we demonstrate an ultracompact diagnostic platform to detect M13 viruses and their mutations from femtoliter volume, sub-100 pM analytes. This platform could pave the way toward an effective diagnosis of diverse pathogens, which is in high demand for handling pandemic situations.

Halide Perovskite Nanocrystal-Enabled Stabilization of Transition Metal Dichalcogenide Nanosheets.

Transition metal dichalcogenide (TMD) nanosheets exfoliated in the liquid phase are of significant interest owing to their potential for scalable and flexible photoelectronic applications. Although various dispersants such as surfactants, oligomers, and polymers are used to obtain highly exfoliated TMD nanosheets, most of them are electrically insulating and need to be removed; otherwise, the photoelectric properties of the TMD nanosheets degrade. Here, inorganic halide perovskite nanocrystals (NCs) of CsPbX3  (X = Cl, Br, or I) are presented as non-destructive dispersants capable of dispersing TMD nanosheets in the liquid phase and enhancing the photodetection properties of the nanosheets, thus eliminating the need to remove the dispersant. MoSe2 nanosheets dispersed in the liquid phase are adsorbed with CsPbCl3  NCs. The CsPbCl3 nanocrystals on MoSe2 efficiently withdraw electrons from the nanosheets, and suppress the dark current of the MoSe2 nanosheets, leading to flexible near-infrared MoSe2  photodetectors with a high ON/OFF photocurrent ratio and detectivity. Moreover, lanthanide ion-doped CsPbCl3  NCs enhance the ON/OFF current ratio to >106 . Meanwhile, the dispersion stability of the MoSe2  nanosheets exfoliated with the perovskite NCs is sufficiently high.

Characterization of Plocamium telfairiae Extract-Functionalized Au Nanostructures and Their Anti-Adipogenic Activity through PLD1.

Here, Au nanostructure (AuNS) biosynthesis was mediated through ethanolic extract of Plocamium telfairiae (PT) without the use of stabilizers or surfactants. PT-functionalized AuNSs (PT-AuNSs) were analyzed using ultraviolet-visible spectroscopy, dynamic light scattering, high-resolution transmission electron microscopy, energy-dispersive spectroscopy, and Fourier-transform infrared spectroscopy. Stable monodisperse PT-AuNSs were synthesized, with a mean size of 15.36 ± 0.10 nm and zeta potential of -35.85 ± 1.36 mV. Moreover, biosynthetic AuNPs with a face-centered structure of PT-AuNS exhibited crystalline characteristics. In addition, many functional groups playing important roles in the biological reduction of PT extracts were adsorbed on the surface of PT-AuNSs. Furthermore, the effects of PT-AuNSs on adipogenesis in immature adipocytes were investigated. PT-AuNSs reduced morphological changes, lowered triglyceride content, and increased lipid accumulation by approximately 78.6% in immature adipocytes compared with the values in mature adipocytes (MDI-induced). PT-AuNS suppressed lipid accumulation by downregulating the transcript and protein expression of C/EBPα, PPARγ, SREBP 1, FAS, and aP2. Finally, PT-AuNS induced the transcript and protein expression of UCP1, PRDM16, and PGC1a, thereby increasing mitochondrial biogenesis in mature adipocytes and effectively inducing brown adipogenesis. In this study, the biosynthesized PT-AuNS was used as a potential therapeutic candidate because it conferred a potent anti-lipogenic effect. As a result, it can be used in various scientific fields such as medicine and the environment.

Cucurbitacin B-, E-, and I-Induced Browning of White Adipocytes Is Promoted by the Inhibition of Phospholipase D2.

The mechanism of white adipose tissue browning is not well understood; however, naturally occurring compounds are known to play a positive role. The effects of cucurbitacins B, E, and I on the browning of mature white adipocytes were investigated. First, the cell viability exhibited by cucurbitacins B, E, and I in pre- and mature adipocytes was verified. Cucurbitacins B, E, and I had no effect on cell viability in pre- and mature adipocytes at concentrations up to 300 nM. To investigate the characteristics of representative beige adipocytes, the formation and morphology of cucurbitacin B, E, and I lipid droplets were verified. The total lipid droplet surface area, maximum Feret diameter, and total Nile red staining intensity of cucurbitacin B-, E-, and I-treated adipocytes were lower than those of mature white adipocytes. Furthermore, treatment of white mature adipocytes with cucurbitacin B, E, and I led to the formation of several small lipid droplets that are readily available for energy expenditure. We evaluated the effect of cucurbitacins B, E, and I on the expression of representative browning markers UCP1, PGC1a, and PRDM16, which participate in the browning of white adipose tissue. Cucurbitacins B, E, and I increased the mRNA and protein expression levels of UCP1, PGC1a, and PRDM16 in a concentration-dependent manner. To promote energy consumption by beige adipocytes, active mitochondrial biogenesis is essential. Next, we investigated the effects of cucurbitacin B, E, and I on mitochondrial biogenesis in mature adipocytes. Mitochondrial mass increased when mature adipocytes were treated with cucurbitacin B, E, and I. The degree of cucurbitacin B-, E- and I-induced transformation of white adipocytes into beige adipocytes was in the order of Cu E > Cu B > Cu I. To verify the effect of phospholipase D2 on the browning of white adipocytes, CAY10594­a PLD2 pharmacological inhibitor, and a knockdown system were used. PLD2 inhibition and knockdown improved the expression levels of UCP1, PGC1a, and PRDM16. In addition, PLD2 inhibition and knockdown in mature white adipocytes promoted mitochondrial biosynthesis. The effect of PLD2 inhibition and knockdown on promoting browning of white adipocytes significantly increased when Cu B, Cu E, and Cu I were co-treated. These data indicate that mature white adipocytes' beige properties were induced by cucurbitacins B, E, and I. These effects became more potent by the inhibition of PLD2. These findings provide a model for determining anti-obesity agents that induce browning and increase energy expenditure in mature white adipocytes.

Fabrication of Chiral M13 Bacteriophage Film by Evaporation-Induced Self-Assembly.

Biomacromolecules are likely to undergo self-assembly and show specific collective behavior concentrated in the medium. Although the assembly procedures have been studied for unraveling their mysteries, there are few cases to directly demonstrate the collective behavior and phase transition process in dynamic systems. In the contribution, the drying process of M13 droplet is investigated, and can be successfully simulated by a doctor blade coating method. The morphologies in the deposited film are measured by atomic force microscopy and the liquid crystal phase development is captured in real time using polarized optical microscope. Collective behaviors near the contact line are characterized by the shape of meniscus curve and particle movement velocity. With considering rheological properties and flow, the resultant chiral film is used to align gold nanorods, and this approach can suggest a way to use M13 bacteriophage as a scaffold for the multi-functional chiral structures.

Enhancement of the response time in organic photorefractive composites using alkoxy-substituted PDCST as a nonlinear optical chromophore: publisher's note.

This publisher's note contains corrections to Opt. Lett.45, 6767 (2020)OPLEDP0146-959210.1364/OL.409743.

Use of Multiple Bacteriophage-Based Structural Color Sensors to Improve Accuracy for Discrimination of Geographical Origins of Agricultural Products.

A single M13 bacteriophage color sensor was previously utilized for discriminating the geographical origins of agricultural products (garlic, onion, and perilla). The resulting discrimination accuracy was acceptable, ranging from 88.6% to 94.0%. To improve the accuracy further, the use of three separate M13 bacteriophage color sensors containing different amino acid residues providing unique individual color changes (Wild sensor: glutamic acid (E)-glycine (G)-aspartic acid (D), WHW sensor: tryptophan (W)-histidine (H)-tryptophan (W), 4E sensor: four repeating glutamic acids (E)) was proposed. This study was driven by the possibility of enhancing sample discrimination by combining mutually characteristic and complimentary RGB signals obtained from each color sensor, which resulted from dissimilar interactions of sample odors with the employed color sensors. When each color sensor was used individually, the discrimination accuracy based on support vector machine (SVM) ranged from 91.8-94.0%, 88.6-90.3%, and 89.8-92.1% for garlic, onion, and perilla samples, respectively. Accuracy improved to 98.0%, 97.5%, and 97.1%, respectively, by integrating all of the RGB signals acquired from the three color sensors. Therefore, the proposed strategy was effective for improving sample discriminability. To further examine the dissimilar responses of each color sensor to odor molecules, typical odor components in the samples (allyl disulfide, allyl methyl disulfide, and perillaldehyde) were measured using each color sensor, and differences in RGB signals were analyzed.

Engineering Efficient Self-Assembled Plasmonic Nanostructures by Configuring Metallic Nanoparticle's Morphology.

We reveal the significance of plasmonic nanoparticle's (NP) shape and its surface morphology en route to an efficient self-assembled plasmonic nanoparticle cluster. A simplified model is simulated in the form of free-space dimer and trimer nanostructures (NPs in the shape of a sphere, cube, and disk). A ~200% to ~125% rise in near-field strength (gap mode enhancement) is observed for spherical NPs in comparison with cubical NPs (from 2 nm to 8 nm gap sizes). Full-width three-quarter maximum reveals better broad-spectral optical performance in a range of ~100 nm (dimer) and ~170 nm (trimer) from spherical NPs as compared to a cube (~60 nm for dimer and trimer). These excellent properties for sphere-based nanostructures are merited from its dipole mode characteristics.

Enhancement of the response time in organic photorefractive composites using alkoxy-substituted PDCST as a nonlinear optical chromophore.

The ease of the molecular orientation of a chromophore has an important effect on the electro-optical (EO) properties of polymeric photorefractive (PR) composites. A derivative of 4-piperidinobenzylidene-malononitrile (PDCST) with an alkoxy group added as a side branch was synthesized to improve the molecular orientation characteristics. Electrophoresis was performed on the polymeric PR composite to which the improved PDCST had been added. The optical properties and response times were examined to evaluate the effects of the substitution of the alkoxy group. PDCST substituted with the alkoxy group showed enhanced EO properties and a PR grating formation rate.

Experimental and numerical evaluation of a genetically engineered M13 bacteriophage with high sensitivity and selectivity for 2,4,6-trinitrotoluene.

Selective and sensitive detection of desired targets is very critical in sensor design. Here, we report a genetically engineered M13 bacteriophage-based sensor system evaluated by quantum mechanics (QM) calculations. Phage display is a facile way to develop the desired peptide sequences, but the resulting sequences can be imperfect peptides for binding of target molecules. A TNT binding peptide (WHW) carrying phage was self-assembled to fabricate thin films and tested for the sensitive and selective surface plasmon resonance-based detection of TNT molecules at the 500 femtomole level. SPR studies performed with the WHW peptide and control peptides (WAW, WHA, AHW) were well-matched with those of the QM calculations. Our combined method between phage engineering and QM calculation will significantly enhance our ability to design selective and sensitive sensors.

Discrimination of phthalate species using a simple phage-based colorimetric sensor in conjunction with hierarchical support vector machine.

Here, the analytical potential of an M13 bacteriophage-based color sensor for discrimination of 4 phthalates with similar molecular structures (bis-(2-ethylhexyl)-phthalate (BEHP), dibutyl phthalate (DBP), diethyl phthalate (DEP), and benzyl-butyl-phthalate (BBP)) was investigated. The pattern and magnitude of the RGB color changes were different depending on the functional groups present in the phthalate structures. For example, BEHP possessing a long alkyl chain resulted in a minute color change, while the variation of color was substantially large when BBP containing an additional benzene ring was measured. Since a tryptophan-histidine-tryptophan residue possessing indole and imidazole was present on the self-assembled phages, the π-π interaction of benzene in BBP with the sensor surface produced a considerably greater color change. To evaluate the multi-modally varying color signals due to diverse interactions of the phthalates with the sensor and to discriminate them, support vector machine (SVM), which can construct a boundary hyperplane among complexly scattered sample groups, was used. In addition, hierarchical SVM (H-SVM) was adopted to deal with multi-class discrimination. The use of H-SVM improved the discrimination accuracy up to 90.1%, compared to 87.1% using SVM. The demonstrated color sensor is versatile and can be potentially adopted as an on-site screening tool. Strategies to improve the accuracy further for real applications are also discussed.

Fast photorefractive response in polymeric composites enabled by the control of chromophore free volume.

The molecular orientation of a chromophore importantly affects the electro-optic characteristics of polymeric photorefractive composites. We designed methyl, ethyl, and isopropyl derivatives of 4-piperidinobenzylidene-malononitrile (PDCST) with the aim of enhancing molecular orientation properties, and investigated the effects of alkyl substitution on the electro-optic properties and response times of polymeric photorefractive composites. The three alkyl-substituted PDCSTs showed enhanced electro-optic responses and photorefractive grating buildup rates.

Graphene-Functionalized Biomimetic Scaffolds for Tissue Regeneration.

Graphene is a two-dimensional atomic layer of graphite, where carbon atoms are assembled in a honeycombed lattice structure. Recently, graphene family nanomaterials, including pristine graphene, graphene oxide and reduced graphene oxide, have increasingly attracted a great deal of interest from researchers in a variety of science, engineering and industrial fields because of their unique structural and functional features. In particular, extensive studies have been actively conducted in the biomedical and related fields, including multidisciplinary and emerging areas, as their stimulating effects on cell behaviors have been becoming an increasing concern. Herein, we are attempting to summarize some of recent findings in the fields of tissue regeneration concerning the graphene family nanomaterial-functionalized biomimetic scaffolds, and to provide the promising perspectives for the possible applications of graphene family nanomaterial.

Graphene-Based Nanocomposites as Promising Options for Hard Tissue Regeneration.

Tissues are often damaged by physical trauma, infection or tumors. A slight injury heals naturally through the normal healing process, while severe injury causes serious health implications. Therefore, many efforts have been devoted to treat and repair various tissue defects. Recently, tissue engineering approaches have attracted a rapidly growing interest in biomedical fields to promote and enhance healing and regeneration of large-scale tissue defects. On the other hand, with the recent advances in nanoscience and nanotechnology, various nanomaterials have been suggested as novel biomaterials. Graphene, a two-dimensional atomic layer of graphite, and its derivatives have recently been found to possess promoting effects on various types of cells. In addition, their unique properties, such as outstanding mechanical and biological properties, allow them to be a promising option for hard tissue regeneration. Herein, we summarized recent research advances in graphene-based nanocomposites for hard tissue regeneration, and highlighted their promising potentials in biomedical and tissue engineering.

Biomimetic self-templating supramolecular structures.

In nature, helical macromolecules such as collagen, chitin and cellulose are critical to the morphogenesis and functionality of various hierarchically structured materials. During tissue formation, these chiral macromolecules are secreted and undergo self-templating assembly, a process whereby multiple kinetic factors influence the assembly of the incoming building blocks to produce non-equilibrium structures. A single macromolecule can form diverse functional structures when self-templated under different conditions. Collagen type I, for instance, forms transparent corneal tissues from orthogonally aligned nematic fibres, distinctively coloured skin tissues from cholesteric phase fibre bundles, and mineralized tissues from hierarchically organized fibres. Nature's self-templated materials surpass the functional and structural complexity achievable by current top-down and bottom-up fabrication methods. However, self-templating has not been thoroughly explored for engineering synthetic materials. Here we demonstrate the biomimetic, self-templating assembly of chiral colloidal particles (M13 phage) into functional materials. A single-step process produces long-range-ordered, supramolecular films showing multiple levels of hierarchical organization and helical twist. Three distinct supramolecular structures are created by this approach: nematic orthogonal twists, cholesteric helical ribbons and smectic helicolidal nanofilaments. Both chiral liquid crystalline phase transitions and competing interfacial forces at the interface are found to be critical factors in determining the morphology of the templated structures during assembly. The resulting materials show distinctive optical and photonic properties, functioning as chiral reflector/filters and structural colour matrices. In addition, M13 phages with genetically incorporated bioactive peptide ligands direct both soft and hard tissue growth in a hierarchically organized manner. Our assembly approach provides insight into the complexities of hierarchical assembly in nature and could be expanded to other chiral molecules to engineer sophisticated functional helical-twisted structures.