Liquid-Liquid Phase Transition Drives Intra-chloroplast Cargo Sorting.

In eukaryotic cells, organelle biogenesis is pivotal for cellular function and cell survival. Chloroplasts are unique organelles with a complex internal membrane network. The mechanisms of the migration of imported nuclear-encoded chloroplast proteins across the crowded stroma to thylakoid membranes are less understood. Here, we identified two Arabidopsis ankyrin-repeat proteins, STT1 and STT2, that specifically mediate sorting of chloroplast twin arginine translocation (cpTat) pathway proteins to thylakoid membranes. STT1 and STT2 form a unique hetero-dimer through interaction of their C-terminal ankyrin domains. Binding of cpTat substrate by N-terminal intrinsically disordered regions of STT complex induces liquid-liquid phase separation. The multivalent nature of STT oligomer is critical for phase separation. STT-Hcf106 interactions reverse phase separation and facilitate cargo targeting and translocation across thylakoid membranes. Thus, the formation of phase-separated droplets emerges as a novel mechanism of intra-chloroplast cargo sorting. Our findings highlight a conserved mechanism of phase separation in regulating organelle biogenesis.

Dominant-negative chaperonin mutation ptCPN60α1S57F uncovers redundancy in chloroplast rRNA processing.

The biogenesis of functional forms of chloroplast ribosomal RNAs (rRNAs) is crucial for the translation of chloroplast mRNAs into polypeptides. However, the molecular mechanisms underlying the proper processing and maturation of chloroplast rRNA species are poorly understood. Through a genetic approach, we isolated and characterized an Arabidopsis mutant, α1-4, harboring a missense mutation in the plastid chaperonin-60α1 gene. Using allelism tests and transgenic manipulation, we determined functional redundancy among ptCPN60 subunits. The ptCPN60α1S57F mutation caused specific defects in the formation of chloroplast rRNA species, including 23S, 5S, and 4.5S rRNAs, but not 16S rRNAs. Allelism tests suggested that the dysfunctional ptCPN60α1S57F competes with other members of the ptCPN60 family. Indeed, overexpression of the ptCPN60α1S57F protein in wild-type plants mimicked the phenotypes observed in the α1-4 mutant, while increasing the endogenous transcriptional levels of ptCPN60α2, ß1, ß2, and ß3 in the α1-4 mutant partially mitigated the abnormal fragmentation processing of chloroplast 23S, 5S, and 4.5S rRNAs. Furthermore, we demonstrated functional redundancy between ptCPN60ß1 and ptCPN60ß2 in chloroplast rRNA processing through double-mutant analysis. Collectively, our data reveal a novel physiological role of ptCPN60 subunits in generating the functional rRNA species of the large 50S ribosomal subunit in Arabidopsis chloroplasts.

The DnaJ proteins DJA6 and DJA5 are essential for chloroplast iron-sulfur cluster biogenesis.

Fe-S clusters are ancient, ubiquitous and highly essential prosthetic groups for numerous fundamental processes of life. The biogenesis of Fe-S clusters is a multistep process including iron acquisition, sulfur mobilization, and cluster formation. Extensive studies have provided deep insights into the mechanism of the latter two assembly steps. However, the mechanism of iron utilization during chloroplast Fe-S cluster biogenesis is still unknown. Here we identified two Arabidopsis DnaJ proteins, DJA6 and DJA5, that can bind iron through their conserved cysteine residues and facilitate iron incorporation into Fe-S clusters by interactions with the SUF (sulfur utilization factor) apparatus through their J domain. Loss of these two proteins causes severe defects in the accumulation of chloroplast Fe-S proteins, a dysfunction of photosynthesis, and a significant intracellular iron overload. Evolutionary analyses revealed that DJA6 and DJA5 are highly conserved in photosynthetic organisms ranging from cyanobacteria to higher plants and share a strong evolutionary relationship with SUFE1, SUFC, and SUFD throughout the green lineage. Thus, our work uncovers a conserved mechanism of iron utilization for chloroplast Fe-S cluster biogenesis.

Arabidopsis pentatricopeptide repeat protein GEND2 participates in mitochondrial RNA editing.

In Arabidopsis, RNA editing alters more than 500 cytidines (C) to uridines (U) in mitochondrial transcripts, a process involving the family of pentatricopeptide repeat (PPR) proteins. Here, we report a previously uncharacterized mitochondrial PLS-type PPR protein, GEND2, which functions in the mitochondrial RNA editing. The T-DNA insertion in the 5'-untranslated region of GEND2, referred to as gend2-1, results in defective root development compared to wild-type (WT) plants. A comprehensive examination of mitochondrial RNA editing sites revealed a significant reduction in the gend2-1 mutant compared to WT plants, affecting six specific mitochondrial RNA editing sites, notably within the mitochondrial genes CcmFn-1, RPSL2 and ORFX. These genes encode critical components of cytochrome protein maturation pathway, mitochondrial ribosomal subunit, and twin arginine translocation subunits, respectively. Further analysis of the transcriptional profile of the gend2-1 mutant and wild type revealed a striking induction of expression in a cluster of genes associated with mitochondrial dysfunction and regulated by ANAC017, a key regulator coordinating organelle functions and stress responses. Intriguingly, the gend2-1 mutation activated an ANAC017-dependent signaling aimed at countering cell wall damage induced by cellulose synthase inhibitors, as well as an ANAC017-independent pathway that retarded root growth under normal condition. Collectively, our findings identify a novel mitochondrial PLS-type PPR protein GEND2, which participates in the editing of six specific mitochondrial RNA editing sites. Furthermore, the gend2-1 mutation triggers two distinct pathways in plants: an ANAC017-dependent pathway and ANAC017-independent pathway.

A case of visual impairment due to HHV-6 encephalitis after allogeneic hematopoietic stem cell transplantation in childhood acute myeloid leukemia-M2 subtype.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a crucial treatment option for children with M2 subtype acute myeloid leukemia (AML). Human herpesvirus 6 (HHV-6) encephalitis following transplantation is a rare postoperative complication, with a poor prognosis and a high fatality rate in allo-HSCT recipients. In this report, a juvenile patient with AMLwas successfully treated after developing visual impairment as a result of HHV-6B encephalitis during allo-HSCT therapy. HHV-6 encephalitis-associated visual impairment after transplantation is rare, and clinical diagnosis and treatment are challenging, requiring more attention in the future.

Dynamics of thin precursor film in wetting of nanopatterned surfaces.

The spreading of a liquid droplet on flat surfaces is a well-understood phenomenon, but little is known about how liquids spread on a rough surface. When the surface roughness is of the nanoscopic length scale, the capillary forces dominate and the liquid droplet spreads by wetting the nanoscale textures that act as capillaries. Here, using a combination of advanced nanofabrication and liquid-phase transmission electron microscopy, we image the wetting of a surface patterned with a dense array of nanopillars of varying heights. Our real-time, high-speed observations reveal that water wets the surface in two stages: 1) an ultrathin precursor water film forms on the surface, and then 2) the capillary action by nanopillars pulls the water, increasing the overall thickness of water film. These direct nanoscale observations capture the previously elusive precursor film, which is a critical intermediate step in wetting of rough surfaces.

Polyunsaturated fatty acids promote the rapid fusion of lipid droplets in Caenorhabditis elegans.

Lipid droplets (LDs) are intracellular organelles that dynamically regulate lipids and energy homeostasis in the cell. LDs can grow through either local lipid synthesis or LD fusion. However, how lipids involving in LD fusion for LD growth is largely unknown. Here, we show that genetic mutation of acox-3 (acyl-CoA oxidase), maoc-1 (enoyl-CoA hydratase), dhs-28 (3-hydroxylacyl-CoA dehydrogenase), and daf-22 (3-ketoacyl-CoA thiolase), all involved in the peroxisomal ß-oxidation pathway in Caenorhabditis elegans, led to rapid fusion of adjacent LDs to form giant LDs (gLDs). Mechanistically, we show that dysfunction of peroxisomal ß-oxidation results in the accumulation of long-chain fatty acid-CoA and phosphocholine, which may activate the sterol-binding protein 1/sterol regulatory element-binding protein to promote gLD formation. Furthermore, we found that inactivation of either FAT-2 (delta-12 desaturase) or FAT-3 and FAT-1 (delta-15 desaturase and delta-6 desaturase, respectively) to block the biosynthesis of polyunsaturated fatty acids (PUFAs) with three or more double bonds (n≥3-PUFAs) fully repressed the formation of gLDs; in contrast, dietary supplementation of n≥3-PUFAs or phosphocholine bearing these PUFAs led to recovery of the formation of gLDs in peroxisomal ß-oxidation-defective worms lacking PUFA biosynthesis. Thus, we conclude that n≥3-PUFAs, distinct from other well-known lipids and proteins, promote rapid LD fusion leading to LD growth.

Purinergic signaling: a potential therapeutic target for depression and chronic pain.

The comorbid mechanism of depression and chronic pain has been a research hotspot in recent years. Until now, the role of purinergic signals in the comorbid mechanism of depression and chronic pain has not been fully understood. This review mainly summarizes the research results published in PubMed during the past 5 years and concludes that purinergic signaling is a potential therapeutic target for comorbid depression and chronic pain, and the purinergic receptors A1, A2A, P2X3, P2X4, and P2X7and P2Y6, P2Y1, and P2Y12 may be important factors. The main potential pathways are as follows: A1 receptor-related G protein-dependent activation of introverted K+ channels (GIRKs), A2A receptor-related effects on the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and MAPK/nuclear factor-κB (NF-κB) pathways, P2X3 receptor-related effects on dorsal root ganglia (DRG) excitability, P2X4 receptor-related effects on proinflammatory cytokines and inflammasome activation, P2X7 receptor-related effects on ion channels, the NLRP3 inflammasome and brain-derived neurotrophic factor (BDNF), and P2Y receptor-related effects on the phospholipase C (PLC)/inositol triphosphate (IP3)/Ca2+ signaling pathway. We hope that the conclusions of this review will provide key ideas for future research on the role of purinergic signaling in the comorbid mechanism of depression and chronic pain.

The multi-faceted mechano-bactericidal mechanism of nanostructured surfaces.

The mechano-bactericidal activity of nanostructured surfaces has become the focus of intensive research toward the development of a new generation of antibacterial surfaces, particularly in the current era of emerging antibiotic resistance. This work demonstrates the effects of an incremental increase of nanopillar height on nanostructure-induced bacterial cell death. We propose that the mechanical lysis of bacterial cells can be influenced by the degree of elasticity and clustering of highly ordered silicon nanopillar arrays. Herein, silicon nanopillar arrays with diameter 35 nm, periodicity 90 nm and increasing heights of 220, 360, and 420 nm were fabricated using deep UV immersion lithography. Nanoarrays of 360-nm-height pillars exhibited the highest degree of bactericidal activity toward both Gram stain-negative Pseudomonas aeruginosa and Gram stain-positive Staphylococcus aureus bacteria, inducing 95 ± 5% and 83 ± 12% cell death, respectively. At heights of 360 nm, increased nanopillar elasticity contributes to the onset of pillar deformation in response to bacterial adhesion to the surface. Theoretical analyses of pillar elasticity confirm that deflection, deformation force, and mechanical energies are more significant for the substrata possessing more flexible pillars. Increased storage and release of mechanical energy may explain the enhanced bactericidal action of these nanopillar arrays toward bacterial cells contacting the surface; however, with further increase of nanopillar height (420 nm), the forces (and tensions) can be partially compensated by irreversible interpillar adhesion that reduces their bactericidal effect. These findings can be used to inform the design of next-generation mechano-responsive surfaces with tuneable bactericidal characteristics for antimicrobial surface technologies.

Ampelopsis grossedentata Represents a New Host of the 16SrI Group of Phytoplasma Associated with Yellow Leaf Symptoms in China.

Ampelopsis grossedentata, commonly known as "Vine Tea" and well-recognized for its rich flavonoid content, is mainly distributed in the southern regions of the Yangtze River basin in China. These regions include Hunan, Hubei, Jiangxi, and Guizhou provinces. Vine Tea is mainly consumed as an herbal tea and has garnered attention for its reported health benefits, including antioxidant, anti-inflammatory, anti-tumor, anti-diabetic, and neuroprotective properties. It has been used to alleviate coughs and sore throats (Zhang et al., 2021; Wang et al., 2017; Gao et al., 2009). In the Zhangjiajie region of Hunan province alone, the Vine Tea planting area reached 7,670.5 hectares and produced commercial goods worth 1.417 billion RMB in 2022. In May 2021, leaf margins and veins fading to yellowing mottling, and crumpling of leaf blades in the shape of a boat symptoms were found in ~16% of Vine Tea plants in the Sanjiakuan Township, Yongding District, Zhangjiajie region (29°15'E, 110°30' N) (Figure 1a, b, c). (Figure 1a, b, c). Phytoplasma-like microbial cells (small oval shaped bacterial cells, around 1000 nm in size) were observed in sieve tube cells in the phloem of diseased leaves using transmission electron microscopy. No such cell was observed in the phloem of healthy leaves (Figure 2a, b). To investigate the potential association between phytoplasma and the observed symptoms of the diseased plants, total DNA was isolated from ten diseasedeaves and compared with ten healthy leaves from the same field using SteadyPure Plant Genomic DNA Extraction Kit. The isolated DNAs were analyzed first in a direct PCR using universal phytoplasma primer pair R16mF2/R16mR1 targeting the 16S rRNA gene (Gundersen and Lee 1996) and specific pair rpF1/rpR1 (Lee et al. 1998) targeting the DNA fragment encoding partial ribosomal proteins (rp) L22 (complete) and S3 and S19 (partial). The initial amplified products were used as templates and further amplified by nested PCR respectively with primer pair R16F2n/R16R2 for the 16S rRNA gene (Lee et al. 1998) and the rpF2/rpR2 primer pair for the rp gene (Martini et al. 2007). No amplification was obtained with DNA from healthy leaf samples using any of the four primer pairs. The amplified fragments from diseased leaves by nested PCR were cloned and sequenced (Qingke Biotech, China). The obtained sequences have been deposited in GenBank with accession numbers OR282806 for the 16S rRNA gene and GenBank OR353012 for the rp gene. BLASTn analysis revealed that the partial 16S rRNA gene sequence in our sample shared 99.4% nucleotide sequence identity with 'Candidatus Phytoplasma sp.' (MW364378) and 'Peony yellows phytoplasma' (KY814723) of the 16SrI group. Similarly, our rp gene sequence shared 99.6% nucleotide identity with the rpI group of phytoplasma such as the 'Balsamine virescence phytoplasma' (JN572890) and 'Paulownia witches'-broom phytoplasma' (HM146079). Phylogenetic analysis of the 16S rRNA and rp sequences using MEGA version 7.0 revealed that the phytoplasma strain associated with A. grossedentata yellow leaf syndrome in our study site belonged to the 16SrI (Candidatus Phytoplasma asteris) group of phytoplasma (Figure 3a, b). Using the interactive online phytoplasma classification tool iPhyClassifier (Zhao et al., 2009), virtual restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene sequences showed our strain having a distinct RFLP map but was closest to that of the onion yellow phytoplasma 16SrI-B subgroup (GenBank accession number: AP006628), with a similarity coefficient of 0.94 (Figure 4a, b). To confirm phytoplasma transmission, healthy plants were inoculated with three scions of infected plants of A. grossedentata. After 16 days, the new leaves of the inoculated A. grossedentata showed yellow leaf symptoms (Figure 5a, b, c), akin to the symptoms originally observed in the field, and the outer contour of the leaf margin appeared chlorotic. After 26 days, primer pairs R16mF2/R16R1 and R16F2n/R16R2 were used for nested PCR detection of phytoplasma in symptomatic A. grossedentata leaves. Phytoplasma was detected in the first and second leaves of symptomatic branches and leaves while negative control showed no amplification. Sequencing of the amplified fragments showed 100% nucleotide identity to the strain from the grafting source. Our results indicated that the pathogen and the disease can be transmitted by tissue grafting, consistent with the biological characteristics of phytoplasma, and further confirmed that the phytoplasma was the pathogen of yellow leaf syndrome of A. grossedentata. Toour knowledge, this is the first report of phytoplasma of group 16SrI affecting A. grossedentata.