SnapShot: Condensates in plant biology.

Plant cells share a number of biological condensates with cells from other eukaryotes. There are, however, a growing number of plant-specific condensates that support different cellular functions. Condensates operating in different plant tissues contribute to aspects of development and stress responses. To view this SnapShot, open or download the PDF.

A CPF-like phosphatase module links transcription termination to chromatin silencing.

The interconnections between co-transcriptional regulation, chromatin environment, and transcriptional output remain poorly understood. Here, we investigate the mechanism underlying RNA 3' processing-mediated Polycomb silencing of Arabidopsis FLOWERING LOCUS C (FLC). We show a requirement for ANTHESIS PROMOTING FACTOR 1 (APRF1), a homolog of yeast Swd2 and human WDR82, known to regulate RNA polymerase II (RNA Pol II) during transcription termination. APRF1 interacts with TYPE ONE SERINE/THREONINE PROTEIN PHOSPHATASE 4 (TOPP4) (yeast Glc7/human PP1) and LUMINIDEPENDENS (LD), the latter showing structural features found in Ref2/PNUTS, all components of the yeast and human phosphatase module of the CPF 3' end-processing machinery. LD has been shown to co-associate in vivo with the histone H3 K4 demethylase FLOWERING LOCUS D (FLD). This work shows how the APRF1/LD-mediated polyadenylation/termination process influences subsequent rounds of transcription by changing the local chromatin environment at FLC.

Biomolecular condensates mediate bending and scission of endosome membranes.

Multivesicular bodies are key endosomal compartments implicated in cellular quality control through their degradation of membrane-bound cargo proteins1-3. The ATP-consuming ESCRT protein machinery mediates the capture and engulfment of membrane-bound cargo proteins through invagination and scission of multivesicular-body membranes to form intraluminal vesicles4,5. Here we report that the plant ESCRT component FREE16 forms liquid-like condensates that associate with membranes to drive intraluminal vesicle formation. We use a minimal physical model, reconstitution experiments and in silico simulations to identify the dynamics of this process and describe intermediate morphologies of nascent intraluminal vesicles. Furthermore, we find that condensate-wetting-induced line tension forces and membrane asymmetries are sufficient to mediate scission of the membrane neck without the ESCRT protein machinery or ATP consumption. Genetic manipulation of the ESCRT pathway in several eukaryotes provides additional evidence for condensate-mediated membrane scission in vivo. We find that the interplay between condensate and machinery-mediated scission mechanisms is indispensable for osmotic stress tolerance in plants. We propose that condensate-mediated scission represents a previously undescribed scission mechanism that depends on the physicomolecular properties of the condensate and is involved in a range of trafficking processes. More generally, FREE1 condensate-mediated membrane scission in multivesicular-body biogenesis highlights the fundamental role of wetting in intracellular dynamics and organization.

Arabidopsis FLL2 promotes liquid-liquid phase separation of polyadenylation complexes.

An important component of cellular biochemistry is the concentration of proteins and nucleic acids in non-membranous compartments1,2. These biomolecular condensates are formed from processes that include liquid-liquid phase separation. The multivalent interactions necessary for liquid-liquid phase separation have been extensively studied in vitro1,3. However, the regulation of this process in vivo is poorly understood. Here we identify an in vivo regulator of liquid-liquid phase separation through a genetic screen targeting factors required for Arabidopsis RNA-binding protein FCA function. FCA contains prion-like domains that phase-separate in vitro, and exhibits behaviour in vivo that is consistent with phase separation. The mutant screen identified a functional requirement for FLL2, a coiled-coil protein, in the formation of FCA nuclear bodies. FCA reduces transcriptional read-through by promoting proximal polyadenylation at many sites in the Arabidopsis genome3,4. FLL2 was required to promote this proximal polyadenylation, but not the binding of FCA to target RNA. Ectopic expression of FLL2 increased the size and number of FCA nuclear bodies. Crosslinking with formaldehyde captured in vivo interactions between FLL2, FCA and the polymerase and nuclease modules of the RNA 3'-end processing machinery. These 3' RNA-processing components colocalized with FCA in the nuclear bodies in vivo, which indicates that FCA nuclear bodies compartmentalize 3'-end processing factors to enhance polyadenylation at specific sites. Our findings show that coiled-coil proteins can promote liquid-liquid phase separation, which expands our understanding of the principles that govern the in vivo dynamics of liquid-like bodies.

Renal-Clearable Organic Probes From D-A-D Type Aza-BODIPY Fluorophores for Multiphoton Deep-Brain Imaging.

Bright near-infrared (NIR) fluorescent probes play an important role in in vivo optical imaging. Here, renal-clearable nanodots prepared from Aza-BODIPY are reported fluorophores for multiphoton brain imaging. The design of donor-acceptor-donor (D-A-D) type conjugated structures endowed the fluorophores with large three-photon absorption cross-section for both 1620 and 2200 nm excitation. The side chain modification and lipid encapsulation yield ultrasmall nanodots (≈4 nm) and a high fluorescence quantum yield (≈0.35) at 720 nm emission in the aqueous phase. The measured three-photon action cross-section of a single Aza-BODIPY fluorophore in the nanodots is ≈30 times higher than the commonly used Sulforhodamine 101 dye. Three-photon deep brain imaging of subcortical structures is demonstrated, reaching a depth of 1900 µm below the brain surface in a live mouse study. The nanodots enabled blood flow measurement at a depth of 1617 µm using line scanning three-photon microscopy (3PM). This work provides superior fluorescent probes for multiphoton deep-brain imaging.

Condensation of SEUSS promotes hyperosmotic stress tolerance in Arabidopsis.

Osmotic stress imposed by drought and high salinity inhibits plant growth and crop yield. However, our current knowledge on the mechanism by which plants sense osmotic stress is still limited. Here, we identify the transcriptional regulator SEUSS (SEU) as a key player in hyperosmotic stress response in Arabidopsis. SEU rapidly coalesces into liquid-like nuclear condensates when extracellular osmolarity increases. The intrinsically disordered region 1 (IDR1) of SEU is responsible for its condensation. IDR1 undergoes conformational changes to adopt more compact states after an increase in molecular crowding both in vitro and in cells, and two predicted α-helical peptides are required. SEU condensation is indispensable for osmotic stress tolerance, and loss of SEU dramatically compromises the expression of stress tolerance genes. Our work uncovers a critical role of biomolecular condensates in cellular stress perception and response and expands our understanding of the osmotic stress pathway.

Efficacy and safety of a cream containing panthenol, prebiotics, and probiotic lysate for improving sensitive skin symptoms.

BACKGROUND: Sensitive skin is a common condition affecting a significant proportion of the population, and there is a growing demand for effective and safe management. AIM: To evaluate the efficacy and safety of a cream containing panthenol, prebiotics, and probiotic lysate as an optimal care for facial sensitive skin. METHODS: A total of 110 participants (64 in group A and 46 in group B) with facial sensitive skin applied the cream twice daily for 28 days. Group A evaluated their sensitive skin, product efficacy, and product use experience at D0 (15 min), D1, D14, and D28. In group B, skin barrier function-related indicators were measured at baseline and on D1, D7, D14, and D28. Dermatologists evaluated tolerance for all participants. RESULTS: After 28 days of use, in group A, 100% of participants reported mildness and comfort with product use. Participants demonstrated significant improvements in skin barrier function-related indicators, including increased stratum corneum moisture content, reduced erythema index, elevated sebum content, decreased trans-epidermal water loss, and diminished skin redness parameter a* value (all p < 0.05). Dermatologist evaluations revealed excellent tolerance among all participants. CONCLUSION: The panthenol-enriched cream with prebiotics and probiotic lysate exhibited substantial clinical efficacy in ameliorating facial sensitive skin conditions, coupled with a high safety profile.

Antagonistic cotranscriptional regulation through ARGONAUTE1 and the THO/TREX complex orchestrates FLC transcriptional output.

Quantitative transcriptional control is essential for physiological and developmental processes in many organisms. Transcriptional output is influenced by cotranscriptional processes interconnected to chromatin regulation, but how the functions of different cotranscriptional regulators are integrated is poorly understood. The Arabidopsis floral repressor locus FLOWERING LOCUS C (FLC) is cotranscriptionally repressed by alternative processing of the antisense transcript COOLAIR. Proximal 3'-end processing of COOLAIR resolves a cotranscriptionally formed R-loop, and this process physically links to a histone-modifying complex FLD/SDG26/LD. This induces a chromatin environment locally that determines low transcription initiation and a slow elongation rate to both sense and antisense strands. Here, we show that ARGONAUTE1 (AGO1) genetically functions in this cotranscriptional repression mechanism. AGO1 associates with COOLAIR and influences COOLAIR splicing dynamics to promote proximal COOLAIR, R-loop resolution, and chromatin silencing. Proteomic analyses revealed physical associations between AGO1, subunits of RNA Polymerase II (Pol II), the splicing-related proteins-the spliceosome NineTeen Complex (NTC) and related proteins (NTR)-and the THO/TREX complex. We connect these activities by demonstrating that the THO/TREX complex activates FLC expression acting antagonistically to AGO1 in COOLAIR processing. Together these data reveal that antagonistic cotranscriptional regulation through AGO1 or THO/TREX influences COOLAIR processing to deliver a local chromatin environment that determines FLC transcriptional output. The involvement of these conserved cotranscriptional regulators suggests similar mechanisms may underpin quantitative transcriptional regulation generally.

Deep Red Light Driven Hydrogen Evolution by Heterojunction Polymer Dots for Diabetic Wound Healing.

We describe small heterojunction polymer dots (Pdots) with deep-red light catalyzed H2 generation for diabetic skin wound healing. The Pdots with donor/acceptor heterojunctions showed remarkably enhanced photocatalytic activity as compared to the donor or acceptor nanoparticles alone. We encapsulate the Pdots and ascorbic acid into liposomes to form Lipo-Pdots nanoreactors, which selectively scavenge ⋅OH radicals in live cells and tissues under 650 nm light illumination. The antioxidant capacity of the heterojunction Pdots is ~10 times higher than that of the single-component Pdots described previously. Under a total light dose of 360 J/cm2, the Lipo-Pdots nanoreactors effectively scavenged ⋅OH radicals and suppressed the expression of pro-inflammatory cytokines in skin tissues, thereby accelerating the healing of skin wounds in diabetic mice. This study provides a feasible solution for safe and effective treatment of diabetic foot ulcers.

The PIF1/PIF3-MED25-HDA19 transcriptional repression complex regulates phytochrome signaling in Arabidopsis.

Light signals are perceived by photoreceptors, triggering the contrasting developmental transition in dark-germinated seedlings. Phytochrome-interacting factors (PIFs) are key regulators of this transition. Despite their prominent functions in transcriptional activation, little is known about PIFs' roles in transcriptional repression. Here, we provide evidence that histone acetylation is involved in regulating phytochrome-PIFs signaling in Arabidopsis. The histone deacetylase HDA19 interacts and forms a complex with PIF1 and PIF3 and the Mediator subunit MED25. The med25/hda19 double mutant mimics and enhances the phenotype of pif1/pif3 in both light and darkness. HDA19 and MED25 are recruited by PIF1/PIF3 to the target loci to reduce histone acetylation and chromatin accessibility, providing a mechanism for PIF1/PIF3-mediated transcriptional repression. Furthermore, MED25 forms liquid-like condensates, which can compartmentalize PIF1/PIF3 and HDA19 in vitro and in vivo, and the number of MED25 puncta increases in darkness. Collectively, our study establishes a mechanism wherein PIF1/PIF3 interact with HDA19 and MED25 to mediate transcriptional repression in the phytochrome signaling pathway and suggests that condensate formation with Mediator may explain the distinct and specific transcriptional activity of PIF proteins.

Site-Specific Modification of Single Domain Antibodies by Enzyme-Immobilized Magnetic Beads.

Nanobodies as imaging agents and drug conjugates have shown great potential for cancer diagnostics and therapeutics. However, site-specific modification of a nanobody with microbial transglutaminase (mTGase) encounters problems in protein separation and purification. Here, we describe a facile yet reliable strategy of immobilizing mTGase onto magnetic beads for site-specific nanobody modification. The mTGase immobilized on magnetic beads (MB-mTGase) exhibits catalytic activity nearly equivalent to that of the free mTGase, with good reusability and universality. Magnetic separation simplifies the protein purification step and reduces the loss of nanobody bioconjugates more effectively than size exclusion chromatography. Using MB-mTGase, we demonstrate site-specific conjugation of nanobodies with fluorescent dyes and polyethylene glycol molecules, enabling targeted immunofluorescence imaging and improved circulation dynamics and tumor accumulation in vivo. The combined advantages of MB-mTGase method, including high conjugation efficiency, quick purification, less protein loss, and recycling use, are promising for site-specific nanobody functionalization and biomedical applications.

Comprehensive assessment of the efficacy and safety of a clay mask in oily and acne skin.

BACKGROUND: Oily skin, characterized by excessive sebum production, can lead to acne and have psychosocial impacts due to changes in appearance. Recent research has shown interest in treatments for oil control, with kaolin and bentonite emerging as promising options. Despite their potential, comprehensive studies on these ingredients are still in the nascent stages. AIM: This study aimed to assess the efficacy of a clay mask (La Roche-Posay Effaclar Sebo-Controlling Mask) in reducing skin oiliness and acne, and its safety for use. METHODS: In this study, 75 adults with oily or combination skin were enrolled and provided with a clay mask for twice-weekly use over 4 weeks. Clinical assessments, using instruments like Sebumeter, Vapometer, and Corneometer, were conducted at baseline, and after 1, 2, and 4 weeks, evaluating acne lesions, skin irritation, sebum content, and skin hydration. Participant self-assessment questionnaires were also utilized for subjective evaluation. Statistical analyses were performed accordingly. RESULTS: The study revealed significant improvements in acne-related outcomes, sebum content, skin evenness, stratum corneum water content, and transepidermal water loss following the application of the clay mask. Pore area and porphyrin area showed no significant changes. Tolerance assessment showed reduced dryness and irritation, with self-assessment indicating high product acceptability and perceived oil control effectiveness. CONCLUSION: This study demonstrated the clay mask's efficacy in managing acne and oily skin, improving hydration and texture. Significant improvements in skin parameters and high product safety were observed, supporting its suitability.

The 3' processing of antisense RNAs physically links to chromatin-based transcriptional control.

Noncoding RNA plays essential roles in transcriptional control and chromatin silencing. At Arabidopsis thaliana FLC, antisense transcription quantitatively influences transcriptional output, but the mechanism by which this occurs is still unclear. Proximal polyadenylation of the antisense transcripts by FCA, an RNA-binding protein that physically interacts with RNA 3' processing factors, reduces FLC transcription. This process genetically requires FLD, a homolog of the H3K4 demethylase LSD1. However, the mechanism linking RNA processing to FLD function had not been established. Here, we show that FLD tightly associates with LUMINIDEPENDENS (LD) and SET DOMAIN GROUP 26 (SDG26) in vivo, and, together, they prevent accumulation of monomethylated H3K4 (H3K4me1) over the FLC gene body. SDG26 interacts with the RNA 3' processing factor FY (WDR33), thus linking activities for proximal polyadenylation of the antisense transcripts to FLD/LD/SDG26-associated H3K4 demethylation. We propose this demethylation antagonizes an active transcription module, thus reducing H3K36me3 accumulation and increasing H3K27me3. Consistent with this view, we show that Polycomb Repressive Complex 2 (PRC2) silencing is genetically required by FCA to repress FLC Overall, our work provides insights into RNA-mediated chromatin silencing.

The Arabidopsis epigenetic regulator ICU11 as an accessory protein of Polycomb Repressive Complex 2.

Molecular mechanisms enabling the switching and maintenance of epigenetic states are not fully understood. Distinct histone modifications are often associated with ON/OFF epigenetic states, but how these states are stably maintained through DNA replication, yet in certain situations switch from one to another remains unclear. Here, we address this problem through identification of Arabidopsis INCURVATA11 (ICU11) as a Polycomb Repressive Complex 2 accessory protein. ICU11 robustly immunoprecipitated in vivo with PRC2 core components and the accessory proteins, EMBRYONIC FLOWER 1 (EMF1), LIKE HETEROCHROMATIN PROTEIN1 (LHP1), and TELOMERE_REPEAT_BINDING FACTORS (TRBs). ICU11 encodes a 2-oxoglutarate-dependent dioxygenase, an activity associated with histone demethylation in other organisms, and mutant plants show defects in multiple aspects of the Arabidopsis epigenome. To investigate its primary molecular function we identified the Arabidopsis FLOWERING LOCUS C (FLC) as a direct target and found icu11 disrupted the cold-induced, Polycomb-mediated silencing underlying vernalization. icu11 prevented reduction in H3K36me3 levels normally seen during the early cold phase, supporting a role for ICU11 in H3K36me3 demethylation. This was coincident with an attenuation of H3K27me3 at the internal nucleation site in FLC, and reduction in H3K27me3 levels across the body of the gene after plants were returned to the warm. Thus, ICU11 is required for the cold-induced epigenetic switching between the mutually exclusive chromatin states at FLC, from the active H3K36me3 state to the silenced H3K27me3 state. These data support the importance of physical coupling of histone modification activities to promote epigenetic switching between opposing chromatin states.

Long-Term In Vivo Glucose Monitoring by Polymer-Dot Transducer in an Injectable Hydrogel Implant.

Optical sensors have attracted a great deal of interest for glucose detection. However, their practical applications for continuous glucose monitoring are still constrained by operational reliability in subcutaneous tissues. Here, we show an implantable hydrogel platform embedded with luminescent polymer dots (Pdots) for sensitive and long-term glucose monitoring. We use Pdot transducer in a polyacrylamide hydrogel matrix to construct an implantable platform. The hydrogel-Pdot transducer showed bright luminescence with ratiometric response to glucose changes. The in vitro and in vivo sensitivities of the hydrogel implant were enhanced by varying the enzyme concentration and injection volume. After implantation, the hydrogel with Pdot transducer remained at the implanted site without migration for 1 month and can be removed from the subcutaneous tissue for further analysis. Our results indicate that the hydrogel-Pdot platform maintains the intrinsic sensing property with excellent stability during 1 month implantation, while fibrous capsule formation on the implant in some cases needs to be solved for long-term continuous glucose monitoring.

Carbon nanotube filter functionalized with MIL-101(Fe) for enhanced flow-through electro-Fenton.

Herein, an electrochemical carbon nanotubes (CNT) filter modified with MIL-101(Fe) has been designed for the electro-Fenton applications by serving as a functional flow-through electrode. Under an electric field, the hybrid filter enabled the in situ generation of H2O2via the two-electron oxygen reduction reaction, which promoted the production of HO by the accelerated Fe2+/Fe3+ cycling of MIL-101(Fe). It was observed that 93.2 ± 1.2% tetracycline and 69.0 ± 0.8% total organic carbon (TOC) were removed in 2 h under the optimized conditions. The electron paramagnetic resonance (EPR) analysis and radical scavenging experiments revealed that HO predominated the tetracycline degradation. As compared to the batch reactor, the performance of the proposed system was improved by 5.6 times owing to the convection-enhanced mass transport. The plausible working mechanism and degradation pathway were also subsequently proposed. The findings reported in this study provide a promising insight for the environmental remediation by integrating nanotechnology and Fenton chemistry.

Coordinated health effects attributable to particulate matter and other pollutants exposures in the North China Plain.

Hebei Province, located in the North China Plain (NCP) and encircling Beijing and Tianjin, has been suffering from severe air pollution. The monthly average fine particulate matter (PM2.5) concentration was up to 276 µg/m3 in Hebei Province, which adversely affects human health. However, few studies evaluated the coordinated health impact of exposure to PM (PM2.5 and PM10) and other key air pollutants (SO2, NO2, CO, and surface ozone (O3)). In this study, we systematically analyzed the health risks (both mortality and morbidity) due to multiple air pollutants exposures in Hebei Province. The economic loss associated with these health consequences was estimated using the value of statistical life (VSL) and cost of illness (COI) methods. Our results show the health burden and economic loss attributable to multiple ambient air pollutants exposures in Hebei Province is substantial. In 2017, the total premature mortality from multiple air pollutants exposures in Hebei Province was 69,833 (95% CI: 55,549-83,028), which was 2.9 times higher than that of the Pearl River Delta region (PRD). Most of the potential economic loss (79.65%) was attributable to premature mortality from air pollution. The total economic loss due to the health consequences of multiple air pollutants exposures was 175.16 (95% CI: 134.61-224.61) billion Chinese Yuan (CNY), which was 4.92% of Hebei Province's annual gross domestic product (GDP). Thus, the adverse health effects and economic loss caused by exposure to multiple air pollutants should be seriously taken into consideration. To alleviate these damages, Hebei's government ought to establish more stringent measures and regulations to better control air pollution.

Intact RNA structurome reveals mRNA structure-mediated regulation of miRNA cleavage in vivo.

MicroRNA (miRNA)-mediated cleavage is involved in numerous essential cellular pathways. miRNAs recognize target RNAs via sequence complementarity. In addition to complementarity, in vitro and in silico studies have suggested that RNA structure may influence the accessibility of mRNAs to miRNA-induced silencing complexes (miRISCs), thereby affecting RNA silencing. However, the regulatory mechanism of mRNA structure in miRNA cleavage remains elusive. We investigated the role of in vivo RNA secondary structure in miRNA cleavage by developing the new CAP-STRUCTURE-seq method to capture the intact mRNA structurome in Arabidopsis thaliana. This approach revealed that miRNA target sites were not structurally accessible for miRISC binding prior to cleavage in vivo. Instead, we found that the unfolding of the target site structure plays a key role in miRISC activity in vivo. We found that the single-strandedness of the two nucleotides immediately downstream of the target site, named Target Adjacent nucleotide Motif, can promote miRNA cleavage but not miRNA binding, thus decoupling target site binding from cleavage. Our findings demonstrate that mRNA structure in vivo can modulate miRNA cleavage, providing evidence of mRNA structure-dependent regulation of biological processes.

NIR-II Fluorescence Imaging Reveals Bone Marrow Retention of Small Polymer Nanoparticles.

The concept that systemically administered nanoparticles are highly accumulated into the liver, spleen and kidney is a central paradigm in the field of nanomedicine. Here, we report that bone is an important organ for retention of small polymer nanoparticles using in vivo fluorescence imaging in the second near-infrared (NIR-II) window. We prepared different sized polymer nanoparticles with both visible and NIR-II fluorescence. NIR-II imaging reveals that the behavior of nanoparticle distribution in bone was largely dependent on the particle size. Small polymer nanoparticles of ∼15 nm diameter showed fast accumulation and long-term retention in bone, while the nanoparticles larger than ∼25 nm were dominantly distributed in liver. Confocal microscopy of bone sections indicated that the nanoparticles were largely distributed in the endothelial cells of sinusoidal vessels in bone marrow. The study provides promising opportunities for bone imaging and treatment of bone-related disease.

Light-Harvesting Fluorescent Spherical Nucleic Acids Self-Assembled from a DNA-Grafted Conjugated Polymer for Amplified Detection of Nucleic Acids.

The ultralow concentration of nucleic acids in complex biological samples requires fluorescence probes with high specificity and sensitivity. Herein, a new kind of spherical nucleic acids (SNAs) is developed by using fluorescent π-conjugated polymers (FCPs) as a light-harvesting antenna to enhance the signal transduction of nucleic acid detection. Specifically, amphiphilic DNA-grafted FCPs are synthesized and self-assemble into FCP-SNA structures. Tuning the hydrophobicity of the graft copolymer can adjust the size and light-harvesting capability of the FCP-SNAs. We observe that more efficient signal amplification occurs in larger FCP-SNAs, as more chromophores are involved, and the energy transfer can go beyond the Förster radius. Accordingly, the optimized FCP-SNA shows an antenna effect of up to 37-fold signal amplification and the limit of detection down to 1.7 pM in microRNA detection. Consequently, the FCP-SNA is applied to amplified in situ nucleic acid detecting and imaging at the single-cell level.