Small molecule activators of TAK1 promotes its activity-dependent ubiquitination and TRAIL-mediated tumor cell death.

TAK1 is a key modulator of both NF-κB signaling and RIPK1. In TNF signaling pathway, activation of TAK1 directly mediates the phosphorylation of IKK complex and RIPK1. In a search for small molecule activators of RIPK1-mediated necroptosis, we found R406/R788, two small molecule analogs that could promote sustained activation of TAK1. Treatment with R406 sensitized cells to TNF-mediated necroptosis and RIPK1-dependent apoptosis by promoting sustained RIPK1 activation. Using click chemistry and multiple biochemical binding assays, we showed that treatment with R406 promotes the activation of TAK1 by directly binding to TAK1, independent of its original target Syk kinase. Treatment with R406 promoted the ubiquitination of TAK1 and the interaction of activated TAK1 with ubiquitinated RIPK1. Finally, we showed that R406/R788 could promote the cancer-killing activities of TRAIL in vitro and in mouse models. Our studies demonstrate the possibility of developing small molecule TAK1 activators to potentiate the effect of TRAIL as anticancer therapies.

Arabidopsis ORP2A mediates ER-autophagosomal membrane contact sites and regulates PI3P in plant autophagy.

Autophagy is an intracellular degradation system for cytoplasmic constituents which is mediated by the formation of a double-membrane organelle termed the autophagosome and its subsequent fusion with the lysosome/vacuole. The formation of the autophagosome requires membrane from the endoplasmic reticulum (ER) and is tightly regulated by a series of autophagy-related (ATG) proteins and lipids. However, how the ER contacts autophagosomes and regulates autophagy remain elusive in plants. In this study, we identified and demonstrated the roles of Arabidopsis oxysterol-binding protein-related protein 2A (ORP2A) in mediating ER-autophagosomal membrane contacts and autophagosome biogenesis. We showed that ORP2A localizes to both ER-plasma membrane contact sites (EPCSs) and autophagosomes, and that ORP2A interacts with both the ER-localized VAMP-associated protein (VAP) 27-1 and ATG8e on the autophagosomes to mediate the membrane contact sites (MCSs). In ORP2A artificial microRNA knockdown (KD) plants, seedlings display retarded growth and impaired autophagy levels. Both ATG1a and ATG8e accumulated and associated with the ER membrane in ORP2A KD lines. Moreover, ORP2A binds multiple phospholipids and shows colocalization with phosphatidylinositol 3-phosphate (PI3P) in vivo. Taken together, ORP2A mediates ER-autophagosomal MCSs and regulates autophagy through PI3P redistribution.

Sequenced-based Rwanda population provides insights into demographic history.

Rwanda is known as the heart of Africa, reflecting the history of the world. Colonization and genocide have led to Rwanda's existing genetic structure. Herein, we used massively parallel sequencing to analyze 296 loci in 185 Rwandans and constructed a database for Rwandan forensic data for the first time. We found the following results: First, forensic parameters demonstrated that all loci were highly informative and could be used for forensic identification and paternity tests in Rwandans. Second, we found that the differences in genetic background between Rwandans and other African populations were similar but slight, as indicated by the massively parallel sequencing panel. Rwandans belonged to the African population and were inseparable from populations from neighboring countries. Also, Rwandans were closer to the European and American populations because of colonization, war, and other reasons. There was no scientific basis for racial classification established by colonization. Further research still needs to be carried out on more loci and larger Rwandan samples.

Photo-Responsive Phase-Separating Fluorescent Molecules for Intracellular Protein Delivery.

Cellular membranes, including the plasma and endosome membranes, are barriers to outside proteins. Various vehicles have been devised to deliver proteins across the plasma membrane, but in many cases, the payload gets trapped in the endosome. Here we designed a photo-responsive phase-separating fluorescent molecule (PPFM) with a molecular weight of 666.8 daltons. The PPFM compound condensates as fluorescent droplets in the aqueous solution by liquid-liquid phase separation (LLPS), which disintegrate upon photoirradiation with a 405 nm light-emitting diode (LED) lamp within 20 min or a 405 nm laser within 3 min. The PPFM coacervates recruit a wide range of peptides and proteins and deliver them into mammalian cells. Photolysis disperses the payload from condensates into the cytosolic space. Altogether, a type of small molecules that are photo-responsive and phase separating are discovered; their coacervates can serve as transmembrane vehicles for intracellular delivery of proteins, whereas photo illumination triggers the cytosolic distribution of the payload.

Phase-Separated Multienzyme Compartmentalization for Terpene Biosynthesis in a Prokaryote.

Liquid-liquid phase separation (LLPS) forms biomolecular condensates or coacervates in cells. Metabolic enzymes can form phase-separated subcellular compartments that enrich enzymes, cofactors, and substrates. Herein, we report the construction of synthetic multienzyme condensates that catalyze the biosynthesis of a terpene, α-farnesene, in the prokaryote E. coli. RGGRGG derived from LAF-1 was used as the scaffold protein to form the condensates by LLPS. Multienzyme condensates were then formed by assembling two enzymes Idi and IspA through an RIAD/RIDD interaction. Multienzyme condensates constructed inside E. coli cells compartmentalized the cytosolic space into regions of high and low enzyme density and led to a significant enhancement of α-farnesene production. This work demonstrates LLPS-driven compartmentalization of the cytosolic space of prokaryotic cells, condensation of a biosynthetic pathway, and enhancement of the biosynthesis of α-farnesene.

A novel missense mutation within the domain of lysine demethylase 4D (KDM4D) gene is strongly associated with testis morphology traits in pigs.

Lysine demethylase 4D (KDM4D) encodes a histone demethylase, which can influence the androgen signaling as well as play an essential role in spermatogenesis. Recently a study has shown that the mRNA of KDM4D is the key to the successful clone of the Macaque monkeys named as "Zhongzhong" and "Huahua," which greatly increased pregnancy rates of female monkey, suggesting that the KDM4D gene may be strongly associated with reproduction. Therefore, the objective of this study was to explore possible single nucleotide polymorphisms (SNPs) within the coding region of KDM4D gene and analyze the associations with testis morphology traits of male pigs. Herein, two SNPs in exon1 of pig KDM4D gene were identified（NC_010451.4:g.1078A > G and NC_010451.4:g.1358G > C）. Among them, the g.1078A > G mutation was located at the JmjN domain which located from 15 to 55 amino acids of KDM4D. Association analyses showed that the g.1078A > G mutation was strongly associated with testis long circumference (TLC), testis short girth (TSG) and testis weight (TW) of Large White (LW) as well as Landrace (LD) (p < 0.05). The AA/AG genotype had a positive effect on testis morphology traits. Briefly, the novel missense mutation g.1078A > G could be a molecular marker for improving testis morphology traits in pig breeding.

Detection of Insertions/Deletions Within SIRT1, SIRT2 and SIRT3 Genes and Their Associations with Body Measurement Traits in Cattle.

Growth traits are complex quantitative traits controlled by numerous candidate genes, and they can be well-evaluated using body measurement traits. As the members of the nicotinamide adenine dinucleotide-dependent family of histone deacetylases, class I sirtuin genes (including SIRT1, SIRT2 and SIRT3) play crucial roles in regulating lipid metabolism, cellular growth and metabolism, suggesting that they are potential candidate genes affecting body measurement traits in animals. Hence, the objective of this work aimed to detect novel insertions/deletions (indels) of SIRT1, SIRT2 and SIRT3 genes in 955 cattle belonging to five breeds, as well as to evaluate their effects on body measurement traits. Herein, the novel 12-bp indel of SIRT1 gene, the 7-bp indel of SIRT2 gene and the 26-bp indel of SIRT3 gene were firstly reported, respectively. The association analysis indicated that the indels within SIRT1 and SIRT2 genes were significantly associated with body measurement traits such as body weight, chest circumference, height at hip cross, hip width, body height, etc. (P < 0.05 or P < 0.01). Therefore, based on these findings, the two novel indel variants within bovine SIRT1 and SIRT2 genes could be considered as potential molecular markers for growth traits in cattle selection practices and breeding.

The effects of aperture position and length in side-vented needles on root canal irrigation: A computational fluid dynamics study.

INTRODUCTION: Root canal irrigation is crucial for infection control during root canal treatment. Side-vented needles for positive pressure irrigation are commonly used in clinical practice. However, variations in needle design among manufacturers can impact the fluid dynamics of irrigation. This study aims to use computational fluid dynamics to explore the flow characteristics of different needle aperture lengths and positions, and their effects on the effectiveness and safety of irrigation, using a validated passive scalar transport numerical model. METHODS: The validation of the CFD irrigant model was achieved by comparing it with an in vitro irrigation experiment model. The CFD model used scalar concentration, while the in vitro experiment model used red dye tracing. Using a standard 30G side-vented needle as a reference, virtual needle models featuring four aperture lengths and three positions were created. These virtual irrigation needles were then placed in two root canal geometries for CFD simulation to evaluate fluid exchange capabilities and related fluid dynamic parameters. RESULTS: The results of the CFD simulation, using a scalar transport model, closely matched the in vitro tracer tests for irrigation experiments across seven root canal geometries. The CFD analysis indicated that positioning the aperture lower increased the irrigant exchange distance. Notably, decreasing the aperture length to 0.25x, and positioning it at the lower end of the needle significantly increased exchange distance and shear stress, while reducing apical pressure. CONCLUSIONS: These results indicate that the position and length of the aperture affect the exchange distance of irrigant flow, wall shear stress, and apical pressure. The CFD validation model for scalar transport, based on a steady state, can function as a valuable tool for optimizing the side-vented needle in research. Further research on the design of side-vented needles will enhance the understanding of flow characteristics beneficial for irrigation efficiency in clinical practice.

Extracellular Vesicle Isolation and Mass Spectrometry-Based Proteomic Analysis in Arabidopsis thaliana.

Extracellular vesicles (EVs) can transport various biological materials, including proteins, lipids, nucleic acids, and metabolites, through the unconventional protein secretion (UPS) pathway. Plant EVs can be classified into at least three major types: tetraspanin 8 (TET8)-positive EVs, penetration 1 (PEN1)-positive EVs, and exocyst-positive organelle (EXPO)-derived EVs. However, the research progress of plant EVs has been hindered due to the limitations inherent in EV isolation techniques. Moreover, since previous research on plant EVs has primarily focused on the interaction between plants and microbes, the biogenesis, transport, and secretion of plant EVs remain unexplored. Recent advances in centrifugation methods for extraction of apoplastic wash fluids, combined with mass spectrometry-based proteomic analysis, provide approaches to identify regulators and cargoes of plant EVs and thus serve as an important step for future studies on the biogenesis and function of plant EVs. Here, we illustrate detailed methods of EV isolation and mass spectrometry-based proteomic analysis in Arabidopsis.

Corrigendum: The possibility of clinical bonding between metal/ceramic brackets to zirconia: in vitro study.

[This corrects the article DOI: 10.3389/fbioe.2024.1354241.].

The possibility of clinical bonding between metal/ceramic brackets to zirconia: in vitro study.

Objective: The present study aimed to assess the bond strength and durability of six bonding agents concerning their application to metal or ceramic brackets and zirconia. Materials and Methods: Six resin cement bonding agents (XT, XTS, RSBU, RGBU, SBPM, and GMP) were chosen for this investigation. Specimens were either stored in distilled water at 37°C for 24 h or subjected to 5,000 thermocycles before conducting a Shear Bond Strength (SBS) test. Statistical analysis of the SBS data was performed using three-way ANOVA and Games-Howell tests (α = 0.05). The Adhesive Remnant Index was examined, and the debonding surface details on brackets and zirconia were observed. Results: For metal brackets, all groups demonstrated clinically acceptable bond strength, irrespective of storage conditions, except for the XT group. Regarding ceramic brackets, all groups displayed acceptable bond strength after 24 h of water storage. However, following thermocycling, a significant decrease in SBS was noted across all groups (p < 0.05), with SBPM exhibiting a higher bond strength. Three-way ANOVA analysis indicated that SBS values were notably influenced by each factor, and an interaction among the three independent variables was observed (p = 0.000). Conclusion: The reliable bond strength between ceramic brackets and zirconia was significantly lower after thermocycling compared to that of metal brackets and zirconia. SBPM exhibited consistent and robust bond strength between ceramic/metal brackets and zirconia across various storage conditions. Furthermore, the HEMA-free adhesive demonstrated a potentially more consistent bonding performance compared to the HEMA-containing adhesive employed in this study.

Novel Association of KLRC4-KLRK1 Gene Polymorphisms with Susceptibility and Progression of Antithyroid Drug-Induced Agranulocytosis.

OBJECTIVE: Antithyroid drug (ATD)-induced agranulocytosis (TIA) is the most serious adverse effect during ATD treatment of Graves' disease (GD). Previously, the MICA gene was reported to be associated with TIA. MICA protein is an important ligand for the NKG2D protein, which is encoded by the KLRK1 gene and KLRC4-KLRK1 read-through transcription. This study further investigated the association between KLRC4-KLRK1 gene polymorphisms and susceptibility to TIA. METHODS: Twenty-eight candidate single nucleotide polymorphisms (SNPs) on KLRC4-KLRK1 read-through transcription were evaluated by the iPLEX MassARRAY system in 209 GD control patients and 38 TIA cases. RESULTS: A significant association of rs2734565 polymorphism with TIA was found (p=0.02, OR=1.80, 95% CI=1.09-2.96). The haplotype C-A-A-C-G, including rs2734565-C, was associated with a significantly higher risk of TIA (p=4.79E-09, OR=8.361, 95% CI=3.737-18.707). In addition, the interval time from hyperthyroidism to agranulocytosis onset was shorter in patients carrying the rs2734565-C allele than in non-carrying groups (45.00 (14.00-6570.00) d vs. 1080.00 (30.00-3600.00) d, p=0.046), and the interval from ATD treatment to agranulocytosis onset was also shorter in patients carrying rs2734565-C allele (29.00 (13.00-75.00) d vs. 57.50 (21.00-240.00) d, p=0.023). CONCLUSIONS: The findings suggest that the KLRC4-KLRK1 gene polymorphism is associated with susceptibility and progression of ATD-induced agranulocytosis. Patients carrying the rs2734565-C allele had a higher susceptibility and faster onset time of TIA.

Using an ER-specific optogenetic mechanostimulator to understand the mechanosensitivity of the endoplasmic reticulum.

The ability of cells to perceive and respond to mechanical cues is essential for numerous biological activities. Emerging evidence indicates important contributions of organelles to cellular mechanosensitivity and mechanotransduction. However, whether and how the endoplasmic reticulum (ER) senses and reacts to mechanical forces remains elusive. To fill the knowledge gap, after developing a light-inducible ER-specific mechanostimulator (LIMER), we identify that mechanostimulation of ER elicits a transient, rapid efflux of Ca2+ from ER in monkey kidney COS-7 cells, which is dependent on the cation channels transient receptor potential cation channel, subfamily V, member 1 (TRPV1) and polycystin-2 (PKD2) in an additive manner. This ER Ca2+ release can be repeatedly stimulated and tuned by varying the intensity and duration of force application. Moreover, ER-specific mechanostimulation inhibits ER-to-Golgi trafficking. Sustained mechanostimuli increase the levels of binding-immunoglobulin protein (BiP) expression and phosphorylated eIF2α, two markers for ER stress. Our results provide direct evidence for ER mechanosensitivity and tight mechanoregulation of ER functions, placing ER as an important player on the intricate map of cellular mechanotransduction.

A condensates-to-VPS41-associated phagic vacuoles conversion pathway controls autophagy degradation in plants.

Autophagy is a universal degradation system in eukaryotic cells. In plants, although autophagosome biogenesis has been extensively studied, the mechanism of how autophagosomes are transported to the vacuole for degradation remains largely unexplored. In this study, we demonstrated that upon autophagy induction, Arabidopsis homotypic fusion and protein sorting (HOPS) subunit VPS41 converts first from condensates to puncta, then to ring-like structures, termed VPS41-associated phagic vacuoles (VAPVs), which enclose autophagy-related gene (ATG)8s for vacuolar degradation. This process is initiated by ADP ribosylation factor (ARF)-like GTPases ARLA1s and occurs concurrently with autophagy progression through coupling with the synaptic-soluble N-ethylmaleimide-sensitive factor attachment protein rmleceptor (SNARE) proteins. Unlike in other eukaryotes, autophagy degradation in Arabidopsis is largely independent of the RAB7 pathway. By contrast, dysfunction in the condensates-to-VAPVs conversion process impairs autophagosome structure and disrupts their vacuolar transport, leading to a significant reduction in autophagic flux and plant survival rate. Our findings suggest that the conversion pathway might be an integral part of the autophagy program unique to plants.

A distinct linker mediating ER-autophagosome membrane contact site (EACS) in plants.

Endoplasmic reticulum (ER) membrane contacts play a central role in regulating autophagosome formation in yeast and mammals. However, a direct functional linkage between the ER and autophagosomes in plants remains elusive. We have recently identified and characterized a plant-unique protein complex consisting of AT4G22540/OSBP2A/ORP2A (oxysterol binding protein-related protein 2A), the ER resident protein AT3G60600/VAP27-1 (vesicle-associated protein 27-1) and AT2G45170/ATG8e (autophagy related 8e) that mediate the ER-autophagosome membrane contact site (EACS) in the model plant Arabidopsis thaliana. Knockdown (KD) of ORP2A affects autophagosome formation and seedling development, whereas ORP2A KD root cells show accumulation of the macroautophagic/autophagic core machinery and PtdIns3P in enlarged ER membranes under autophagy conditions. This study reveals the molecular architecture and functions of a distinct plant EACS in regulating autophagosome formation via lipid redistribution.

Transcriptome-wide association study by different approaches reveals candidate causal genes for cannabis use disorder.

Cannabis is one of the most commonly used psychoactive substances, which could induce moderate-severe cannabis use disorders (CUD). Here, a tissue-specific transcriptome-wide association study (TWAS) of CUD was performed by FUSION and S-PrediXcan, utilizing a genome-wide association study (GWAS) dataset of CUD (including 43,380 cases and 141,385 controls of European ancestry) and gene expression reference data from 17 different brain-related and non-brain related tissues, with totally 26 TWAS-associated genes were identified, including CADM2 (P = 2.13 × 10-17), SRR (P = 8.09 × 10-9) and TUFM (P = 1.24 × 10-8). Fine-mapping of causal gene sets (FOCUS) was used to prioritize genes with strong evidence for causality, and SRR, CADM2-AS1, and SH2B1 were prioritized with a posterior probability of 0.973, 0.951, and 0.788, respectively. Furthermore, gene ontology (GO) and pathway enrichment analysis on CUD-associated genes were performed, including cytosol, protein binding, nucleoplasm, metabolic pathways, and herpes simplex virus 1 infection. These findings could provide new insights for understanding the mechanism of CUD.

VAMP724 and VAMP726 are involved in autophagosome formation in Arabidopsis thaliana.

Macroautophagy/autophagy, an evolutionarily conserved degradative process essential for cell homeostasis and development in eukaryotes, involves autophagosome formation and fusion with a lysosome/vacuole. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play important roles in regulating autophagy in mammals and yeast, but relatively little is known about SNARE function in plant autophagy. Here we identified and characterized two Arabidopsis SNAREs, AT4G15780/VAMP724 and AT1G04760/VAMP726, involved in plant autophagy. Phenotypic analysis showed that mutants of VAMP724 and VAMP726 are sensitive to nutrient-starved conditions. Live-cell imaging on mutants of VAMP724 and VAMP726 expressing YFP-ATG8e showed the formation of abnormal autophagic structures outside the vacuoles and compromised autophagic flux. Further immunogold transmission electron microscopy and electron tomography (ET) analysis demonstrated a direct connection between the tubular autophagic structures and the endoplasmic reticulum (ER) in vamp724-1 vamp726-1 double mutants. Further transient co-expression, co-immunoprecipitation and double immunogold TEM analysis showed that ATG9 (autophagy related 9) interacts and colocalizes with VAMP724 and VAMP726 in ATG9-positive vesicles during autophagosome formation. Taken together, VAMP724 and VAMP726 regulate autophagosome formation likely working together with ATG9 in Arabidopsis.Abbreviations: ATG, autophagy related; BTH, benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester; Conc A, concanamycin A; EM, electron microscopy; ER, endoplasmic reticulum; FRET, Förster/fluorescence resonance energy transfer; MS, Murashige and Skoog; MVB, multivesicular body; PAS, phagophore assembly site; PM, plasma membrane; PVC, prevacuolar compartment; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TEM, transmission electron microscopy; TGN, trans-Golgi network; WT, wild-type.

The plant unique ESCRT component FREE1 regulates autophagosome closure.

The energy sensor AMP-activated protein kinase (AMPK) can activate autophagy when cellular energy production becomes compromised. However, the degree to which nutrient sensing impinges on the autophagosome closure remains unknown. Here, we provide the mechanism underlying a plant unique protein FREE1, upon autophagy-induced SnRK1α1-mediated phosphorylation, functions as a linkage between ATG conjugation system and ESCRT machinery to regulate the autophagosome closure upon nutrient deprivation. Using high-resolution microscopy, 3D-electron tomography, and protease protection assay, we showed that unclosed autophagosomes accumulated in free1 mutants. Proteomic, cellular and biochemical analysis revealed the mechanistic connection between FREE1 and the ATG conjugation system/ESCRT-III complex in regulating autophagosome closure. Mass spectrometry analysis showed that the evolutionary conserved plant energy sensor SnRK1α1 phosphorylates FREE1 and recruits it to the autophagosomes to promote closure. Mutagenesis of the phosphorylation site on FREE1 caused the autophagosome closure failure. Our findings unveil how cellular energy sensing pathways regulate autophagosome closure to maintain cellular homeostasis.

Shear bond strength of different bonding agents to orthodontic metal bracket and zirconia.

This study aimed to evaluate the shear bond strength (SBS) of four bonding agents used to bond metal brackets to zirconia under different storage conditions. Four bonding agents were used [FLC: (Fuji ORTHO LC), XT: (TransbondTM XT), RUC-SBU: (Rely XTM Ultimate Clicker Adhesive Resin Cement+Single Bond Universal), and RUC-GBU: (Rely XTM Ultimate Clicker Adhesive Resin Cement+Gluma Bond Universal)] to bond two types of metal brackets (PT/3M) to zirconia surfaces, and they were stored in water at 37ºC for 24 h or thermocycling for 3,000 cycles. The SBS data of RUC-SBU and RUC-GBU using PT brackets were significantly higher than those of 3M brackets before and after thermocycling. It could be concluded that RUC-SBU and RUC-GBU could offer sufficient bond strength between metal brackets and zirconia for the short term compared with FLC and XT. The design of brackets can significantly affect the bond strength to zirconia.