TNIP1 inhibits selective autophagy via bipartite interaction with LC3/GABARAP and TAX1BP1.

Mitophagy is a form of selective autophagy that disposes of superfluous and potentially damage-inducing organelles in a tightly controlled manner. While the machinery involved in mitophagy induction is well known, the regulation of the components is less clear. Here, we demonstrate that TNIP1 knockout in HeLa cells accelerates mitophagy rates and that ectopic TNIP1 negatively regulates the rate of mitophagy. These functions of TNIP1 depend on an evolutionarily conserved LIR motif as well as an AHD3 domain, which are required for binding to the LC3/GABARAP family of proteins and the autophagy receptor TAX1BP1, respectively. We further show that phosphorylation appears to regulate its association with the ULK1 complex member FIP200, allowing TNIP1 to compete with autophagy receptors, which provides a molecular rationale for its inhibitory function during mitophagy. Taken together, our findings describe TNIP1 as a negative regulator of mitophagy that acts at the early steps of autophagosome biogenesis.

Nix interacts with WIPI2 to induce mitophagy.

Nix is a membrane-anchored outer mitochondrial protein that induces mitophagy. While Nix has an LC3-interacting (LIR) motif that binds to ATG8 proteins, it also contains a minimal essential region (MER) that induces mitophagy through an unknown mechanism. We used chemically induced dimerization (CID) to probe the mechanism of Nix-mediated mitophagy and found that both the LIR and MER are required for robust mitophagy. We find that the Nix MER interacts with the autophagy effector WIPI2 and recruits WIPI2 to mitochondria. The Nix LIR motif is also required for robust mitophagy and converts a homogeneous WIPI2 distribution on the surface of the mitochondria into puncta, even in the absence of ATG8s. Together, this work reveals unanticipated mechanisms in Nix-induced mitophagy and the elusive role of the MER, while also describing an interesting example of autophagy induction that acts downstream of the canonical initiation complexes.

Reciprocal Roles of Tom7 and OMA1 during Mitochondrial Import and Activation of PINK1.

Mutations in PTEN-induced kinase 1 (PINK1) can cause recessive early-onset Parkinson's disease (PD). Import arrest results in PINK1 kinase activation specifically on damaged mitochondria, triggering Parkin-mediated mitophagy. Here, we show that PINK1 import is less dependent on Tim23 than on mitochondrial membrane potential (ΔΨm). We identified a negatively charged amino acid cluster motif that is evolutionarily conserved just C-terminal to the PINK1 transmembrane. PINK1 that fails to accumulate at the outer mitochondrial membrane, either by mutagenesis of this negatively charged motif or by deletion of Tom7, is imported into depolarized mitochondria and cleaved by the OMA1 protease. Some PD patient mutations also are defective in import arrest and are rescued by the suppression of OMA1, providing a new potential druggable target for PD. These results suggest that ΔΨm loss-dependent PINK1 import arrest does not result solely from Tim23 inactivation but also through an actively regulated "tug of war" between Tom7 and OMA1.

Spatiotemporal Control of ULK1 Activation by NDP52 and TBK1 during Selective Autophagy.

Selective autophagy recycles damaged organelles and clears intracellular pathogens to prevent their aberrant accumulation. How ULK1 kinase is targeted and activated during selective autophagic events remains to be elucidated. In this study, we used chemically inducible dimerization (CID) assays in tandem with CRISPR KO lines to systematically analyze the molecular basis of selective autophagosome biogenesis. We demonstrate that ectopic placement of NDP52 on mitochondria or peroxisomes is sufficient to initiate selective autophagy by focally localizing and activating the ULK1 complex. The capability of NDP52 to induce mitophagy is dependent on its interaction with the FIP200/ULK1 complex, which is facilitated by TBK1. Ectopically tethering ULK1 to cargo bypasses the requirement for autophagy receptors and TBK1. Focal activation of ULK1 occurs independently of AMPK and mTOR. Our findings provide a parsimonious model of selective autophagy, which highlights the coordination of ULK1 complex localization by autophagy receptors and TBK1 as principal drivers of targeted autophagosome biogenesis.

Mitochondria and peroxisomes are NIXed for clearance.

A recent report shows that the iron chelator DFP induces both mitophagy and pexophagy in a BNIP3/NIX-dependent manner. Previously known as a mitophagy receptor, NIX was also independently localized to peroxisomes to promote pexophagy in several physiological conditions, illustrating the significance of this novel function.

Bcl-x(L) retrotranslocates Bax from the mitochondria into the cytosol.

The Bcl-2 family member Bax translocates from the cytosol to mitochondria, where it oligomerizes and permeabilizes the mitochondrial outer membrane to promote apoptosis. Bax activity is counteracted by prosurvival Bcl-2 proteins, but how they inhibit Bax remains controversial because they neither colocalize nor form stable complexes with Bax. We constrained Bax in its native cytosolic conformation within cells using intramolecular disulfide tethers. Bax tethers disrupt interaction with Bcl-x(L) in detergents and cell-free MOMP activity but unexpectedly induce Bax accumulation on mitochondria. Fluorescence loss in photobleaching (FLIP) reveals constant retrotranslocation of WT Bax, but not tethered Bax, from the mitochondria into the cytoplasm of healthy cells. Bax retrotranslocation depends on prosurvival Bcl-2 family proteins, and inhibition of retrotranslocation correlates with Bax accumulation on the mitochondria. We propose that Bcl-x(L) inhibits and maintains Bax in the cytosol by constant retrotranslocation of mitochondrial Bax.

The implication of long non-coding RNA expression profile in rheumatoid arthritis: Correlation with treatment response to tumor necrosis factor inhibitor.

OBJECTIVE: This study aimed to investigate the linkage of long non-coding RNA (lncRNA) expression profile with etanercept response in rheumatoid arthritis (RA) patients. METHODS: Peripheral blood mononuclear cell (PBMC) samples were collected from 80 RA patients prior to etanercept treatment. Samples from eight responders and eight non-responders at week 24 (W24) were proposed to RNA-sequencing, then 10 candidate lncRNAs were sorted and their PBMC expressions were validated by reverse transcription quantitative chain reaction (RT-qPCR) in 80 RA patients. Subsequently, clinical response by lncRNA (CRLnc) prediction model was established. RESULTS: RNA-sequencing identified 254 up-regulated and 265 down-regulated lncRNAs in W24 responders compared with non-responders, which were enriched in immune or joint related pathways such as B-cell receptor signaling, osteoclast differentiation and T-cell receptor signaling pathways, etc. By reverse transcription quantitative chain reaction (RT-qPCR) validation: Two lncRNAs were correlated with W4 response, three lncRNAs were correlated with W12 response, seven lncRNAs were correlated with W24 response. Subsequently, to construct and validate CRLnc prediction model, 80 RA patients were randomly divided into test set (n = 40) and validation set (n = 40). In the test set, lncRNA RP3-466P17.2 (OR = 9.743, P = .028), RP11-20D14.6 (OR = 10.935, P = .007), RP11-844P9.2 (OR = 0.075, P = .022), and TAS2R64P (OR = 0.044, P = .016) independently related to W24 etanercept response; then CRLnc prediction model integrating these four lncRNAs presented a good value in predicting W24 etanercept response (Area Under Curve (AUC): 0.956, 95%CI: 0.896-1.000). However, in the validation set, the CRLnc prediction model only exhibited a certain value in predicting W24 etanercept response (AUC: 0.753, 95%CI: 0.536-0.969). CONCLUSIONS: CRLnc prediction model is potentially a useful tool to instruct etanercept treatment in RA patients.

Ultrasound-guided totally implantable venous access ports placement via right brachiocephalic vein in pediatric population: A clinical debut.

BACKGROUND AND OBJECTIVES: To investigate the feasibility and safety of ultrasound-guided totally implantable venous access ports (TIVAPs) via the right brachiocephalic vein (BCV) in pediatric patients. METHODS: A single-institute retrospective review was performed on 35 pediatric patients with predominantly hematological malignancies (88.6%) who underwent TIVAP implantation via ultrasound-guided right BCV approach from July 2018 to June 2021. The catheter tip was adjusted to be positioned at the cavoatrial junction under pulsed fluoroscopic guidance. Technical success rate, procedural information, and TIVAP-related complications were evaluated. RESULTS: All the pediatric TIVAP devices were successfully implanted via right BCV access. Venous access was successful by first attempt in 32 children (91%), two cases (5.7%) required a second attempt, and one patient (2.9%) required a third attempt. The mean procedural time was 44.6 ± 6.4 minutes (range: 34-62 minutes). No intraoperative complications occurred. The average TIVAP indwelling time was 564 ± 208 days (range: 193-1014 days), with a cumulative 19,723 catheter-days. Overall, three patients (8.6%) experienced four postoperative complications (two cases of local hematoma and two catheter dysfunctions) at a rate of 0.2 per 1000 catheter-days. No other complications such as wound dehiscence, delayed incision healing, catheter-related thrombosis (CRT), catheter malposition/fracture, surgical site infection, catheter-related bloodstream infection (CRBSI), pinch-off syndrome, and drug extravasation were observed during follow-up. CONCLUSIONS: Ultrasound-guided right BCV access for TIVAP placement in pediatric patients appears to be technically feasible, safe, and effective. Further large-sample, prospective studies are warranted.

Nanomaterials and nanotechnology for the delivery of agrochemicals: strategies towards sustainable agriculture.

Nanomaterials (NMs) have received considerable attention in the field of agrochemicals due to their special properties, such as small particle size, surface structure, solubility and chemical composition. The application of NMs and nanotechnology in agrochemicals dramatically overcomes the defects of conventional agrochemicals, including low bioavailability, easy photolysis, and organic solvent pollution, etc. In this review, we describe advances in the application of NMs in chemical pesticides and fertilizers, which are the two earliest and most researched areas of NMs in agrochemicals. Besides, this article concerns with the new applications of NMs in other agrochemicals, such as bio-pesticides, nucleic acid pesticides, plant growth regulators (PGRs), and pheromone. We also discuss challenges and the industrialization trend of NMs in the field of agrochemicals. Constructing nano-agrochemical delivery system via NMs and nanotechnology facilitates the improvement of the stability and dispersion of active ingredients, promotes the precise delivery of agrochemicals, reduces residual pollution and decreases labor cost in different application scenarios, which is potential to maintain the sustainability of agricultural systems and improve food security by increasing the efficacy of agricultural inputs.

Nano Functional Food: Opportunities, Development, and Future Perspectives.

A functional food is a kind of food with special physiological effects that can improve health status or reduce illness. However, the active ingredients in functional foods are usually very low due to the instability and easy degradation of some nutrients. Therefore, improving the utilization rate of the effective ingredients in functional food has become the key problem. Nanomaterials have been widely used and studied in many fields due to their small size effect, high specific surface area, high target activity, and other characteristics. Therefore, it is a feasible method to process and modify functional food using nanotechnology. In this review, we summarize the nanoparticle delivery system and the food nanotechnology in the field of functional food. We also summarize and prospect the application, basic principle, and latest development of nano-functional food and put forward corresponding views.

Versatile Oral Insulin Delivery Nanosystems: From Materials to Nanostructures.

Diabetes is a chronic metabolic disease characterized by lack of insulin in the body leading to failure of blood glucose regulation. Diabetes patients usually need frequent insulin injections to maintain normal blood glucose levels, which is a painful administration manner. Long-term drug injection brings great physical and psychological burden to diabetic patients. In order to improve the adaptability of patients to use insulin and reduce the pain caused by injection, the development of oral insulin formulations is currently a hot and difficult topic in the field of medicine and pharmacy. Thus, oral insulin delivery is a promising and convenient administration method to relieve the patients. However, insulin as a peptide drug is prone to be degraded by digestive enzymes. In addition, insulin has strong hydrophilicity and large molecular weight and extremely low oral bioavailability. To solve these problems in clinical practice, the oral insulin delivery nanosystems were designed and constructed by rational combination of various nanomaterials and nanotechnology. Such oral nanosystems have the advantages of strong adaptability, small size, convenient processing, long-lasting pharmaceutical activity, and drug controlled-release, so it can effectively improve the oral bioavailability and efficacy of insulin. This review summarizes the basic principles and recent progress in oral delivery nanosystems for insulin, including physiological absorption barrier of oral insulin and the development of materials to nanostructures for oral insulin delivery nanosystems.

Construction and Characterization of Novel Hydrophilic Nanospheres Loaded with Lambda-Cyhalothrin via Ultrasonic Emulsification-Solvent Evaporation.

Safe and efficient pesticide formulations have attracted great attention for the prevention and control of diseases and pests. In recent years, improving the effectiveness and duration of pesticides through nanotechnology has become a research hotspot in the field of pesticide formulations. Here, we develop a novel hydrophilic lambda-cyhalothrin nanospheres encapsulated with poly(styrene-co-maleic anhydride) (PSMA) via the ultrasonic emulsification-solvent evaporation method, which exhibited better particle size uniformity and dispersion in comparison with the traditional method. The effects of PSMA content, oil phase/water phase ratio and phacoemulsification time on the particle size and morphology of nanoparticles were investigated to optimize preparation process parameters. Meanwhile, the wettability and adhesion behavior on the leaf surface, the release properties, and the storage stability of nanoparticles were characterized to evaluate the performance of the novel nano-formulation. This work not only establishes a facile and promising method for the applicable of insoluble pesticides, but also develops an innovative nano-formulation with hydrophilicity and high leaf adhesion, which opens a new direction in plant protection and residue reduction.

Fluorescence-based ATG8 sensors monitor localization and function of LC3/GABARAP proteins.

Autophagy is a cellular surveillance pathway that balances metabolic and energy resources and transports specific cargos, including damaged mitochondria, other broken organelles, or pathogens for degradation to the lysosome. Central components of autophagosomal biogenesis are six members of the LC3 and GABARAP family of ubiquitin-like proteins (mATG8s). We used phage display to isolate peptides that possess bona fide LIR (LC3-interacting region) properties and are selective for individual mATG8 isoforms. Sensitivity of the developed sensors was optimized by multiplication, charge distribution, and fusion with a membrane recruitment (FYVE) or an oligomerization (PB1) domain. We demonstrate the use of the engineered peptides as intracellular sensors that recognize specifically GABARAP, GABL1, GABL2, and LC3C, as well as a bispecific sensor for LC3A and LC3B. By using an LC3C-specific sensor, we were able to monitor recruitment of endogenous LC3C to Salmonella during xenophagy, as well as to mitochondria during mitophagy. The sensors are general tools to monitor the fate of mATG8s and will be valuable in decoding the biological functions of the individual LC3/GABARAPs.

The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy.

Protein aggregates and damaged organelles are tagged with ubiquitin chains to trigger selective autophagy. To initiate mitophagy, the ubiquitin kinase PINK1 phosphorylates ubiquitin to activate the ubiquitin ligase parkin, which builds ubiquitin chains on mitochondrial outer membrane proteins, where they act to recruit autophagy receptors. Using genome editing to knockout five autophagy receptors in HeLa cells, here we show that two receptors previously linked to xenophagy, NDP52 and optineurin, are the primary receptors for PINK1- and parkin-mediated mitophagy. PINK1 recruits NDP52 and optineurin, but not p62, to mitochondria to activate mitophagy directly, independently of parkin. Once recruited to mitochondria, NDP52 and optineurin recruit the autophagy factors ULK1, DFCP1 and WIPI1 to focal spots proximal to mitochondria, revealing a function for these autophagy receptors upstream of LC3. This supports a new model in which PINK1-generated phospho-ubiquitin serves as the autophagy signal on mitochondria, and parkin then acts to amplify this signal. This work also suggests direct and broader roles for ubiquitin phosphorylation in other autophagy pathways.

The Development of Novel N-Terminal Pro-Peptide of Type I Chemiluminescence Assay.

BACKGROUND: P1NP can be used for monitoring patients treated with both bisphosphonates and teriparatide as bone formation markers. P1NP assays include two types, intact trimeric form of P1NP assay and total P1NP assay. In this study we provided another type of P1NP assay. METHODS: The α-1 chain was constructed as recombined P1NP protein in the Corynebacterium glutamicum gene expression system. Native proteins were purified from Hydrothorax. Antibody clones were screened using mice immune to the α-1 chain peptide. The screened antibody was used for assay development. Assay performance was verified and afterwards the method comparison was analyzed between the self-developed assay and Roche P1NP assay. RESULTS: α-1 chain and native P1NP proteins were purified and used for antibody selection and making the calibrator. Three clones of antibody were screened and 2 of them were used in the assay development. The assay performance was characterized, including the linearity, precision, and sensitivity. Method comparison was also performed between our assay and Roche P1NP assay showing a 0.98 slope. CONCLUSIONS: A new P1NP assay was provided that recognizes only the α-1 chain and, thus, may provide more insight for disease monitoring when the P1NP assay is applied in clinic in the future.

Study of microRNA Expression Profile in Different Regions of Ram Epididymis.

The regional expression of epididymal genes provides a guarantee for sperm maturation. As a class of endogenous non-coding small RNAs, microRNAs (miRNAs) play an important role in spermatogenesis, maturation and fertilization. Currently, the regulatory role of miRNA in the epididymis is poorly understood. Here, transcriptome sequencing was used to analyse miRNA expression profiles in three regions of the epididymis of rams, including caput, corpus and cauda. The results showed that there were 13 known miRNAs between the caput and corpus controls, 29 between the caput and cauda and 22 differences between the corpus and cauda. Based on the analysis of miRNA target genes by GO and KEGG, a negative regulation network of miRNA-mRNA was constructed in which let-7, miR-541-5p, miR-133b and miR-150 may play an important regulatory role in the maturation regulation of ram epididymal sperm. This research provides a reference for studying the regulation mechanism of sperm maturation in male epididymis and improving semen quality and male reproductive performance.

Preparation and Size Control of Efficient and Safe Nanopesticides by Anodic Aluminum Oxide Templates-Assisted Method.

Efficient and safe nanopesticides play an important role in pest control due to enhancing target efficiency and reducing undesirable side effects, which has become a hot spot in pesticide formulation research. However, the preparation methods of nanopesticides are facing critical challenges including low productivity, uneven particle size and batch differences. Here, we successfully developed a novel, versatile and tunable strategy for preparing buprofezin nanoparticles with tunable size via anodic aluminum oxide (AAO) template-assisted method, which exhibited better reproducibility and homogeneity comparing with the traditional method. The storage stability of nanoparticles at different temperatures was evaluated, and the release properties were also determined to evaluate the performance of nanoparticles. Moreover, the present method is further demonstrated to be easily applicable for insoluble drugs and be extended for the study of the physicochemical properties of drug particles with different sizes.

A Rapid Chemiluminescence Immunoassay for Total Vitamin D Status Assessment in Fingertip Blood.

BACKGROUND: Almost all the automated chemiluminescence analyzers in the market cannot detect total 25-hy-droxyvitamin D (25-OH VD) in fingertip blood sample due to the limited volume. This study aimed to develop a relatively simple pretreatment method to obtain plasma from the fingertip blood samples with limited volume and to establish and evaluate the performance of a chemiluminescence immunoassay (CLIA) for detecting 25-OH VD in plasma. METHODS: Fixed volume of plasma was obtained using a special glass capillary to pretreat the fingertip blood. The coated microwells were inserted and the reagents were dispensed into wells in the test strips which can be continuously tested on a small automated chemiluminescence analyzer. The performance of the developed method was evaluated, and the method comparison studies were conducted. RESULTS: The limit of detection (LOD) was 1.38 ng/mL. The intra-assay precision of the anticoagulant whole blood samples with low, medium, high concentrations of 25-OH VD were 5.45%, 8.20%, 4.97%, respectively. Different metabolic forms of vitamin D have different degrees of cross-interference with antibodies used in the developed method, especially the active metabolites. The cross reactivity (CR) values of 1,25-dihydroxyvitamin D2 (1,25-(OH)2 VD2) and 1,25-dihydroxyvitamin D3 (1,25-(OH)2 VD3) were 5.83% and 9.13%, respectively. The method comparison results of plasma from the venous blood samples showed that correlation coefficient (R2) is 0.94, with a slight positive bias of +1.1% [95% limits of agreement (± 1.96 SD); -25.8% to 28.1%]. Comparing the results detected by the developed method (plasma from fingertip blood samples) with Roche (plasma from anticoagulant venous blood), good linear relativity was noted, with a slight negative bias of -1.2% [95% limits of agreement (± 1.96 SD); -28.7% to 26.4%]. CONCLUSIONS: The developed pretreatment method for fingertip blood and the CLIA method can be used to assess the vitamin D status. The correlation of performance and method with the Roche test was good, and the whole test needs only 30 minutes. The developed method can fully satisfy very busy clinical labs.

High-content genome-wide RNAi screens identify regulators of parkin upstream of mitophagy.

An increasing body of evidence points to mitochondrial dysfunction as a contributor to the molecular pathogenesis of neurodegenerative diseases such as Parkinson's disease. Recent studies of the Parkinson's disease associated genes PINK1 (ref. 2) and parkin (PARK2, ref. 3) indicate that they may act in a quality control pathway preventing the accumulation of dysfunctional mitochondria. Here we elucidate regulators that have an impact on parkin translocation to damaged mitochondria with genome-wide small interfering RNA (siRNA) screens coupled to high-content microscopy. Screening yielded gene candidates involved in diverse cellular processes that were subsequently validated in low-throughput assays. This led to characterization of TOMM7 as essential for stabilizing PINK1 on the outer mitochondrial membrane following mitochondrial damage. We also discovered that HSPA1L (HSP70 family member) and BAG4 have mutually opposing roles in the regulation of parkin translocation. The screens revealed that SIAH3, found to localize to mitochondria, inhibits PINK1 accumulation after mitochondrial insult, reducing parkin translocation. Overall, our screens provide a rich resource to understand mitochondrial quality control.

Phosphorylation of OPTN by TBK1 enhances its binding to Ub chains and promotes selective autophagy of damaged mitochondria.

Selective autophagy of damaged mitochondria requires autophagy receptors optineurin (OPTN), NDP52 (CALCOCO2), TAX1BP1, and p62 (SQSTM1) linking ubiquitinated cargo to autophagic membranes. By using quantitative proteomics, we show that Tank-binding kinase 1 (TBK1) phosphorylates all four receptors on several autophagy-relevant sites, including the ubiquitin- and LC3-binding domains of OPTN and p62/SQSTM1 as well as the SKICH domains of NDP52 and TAX1BP1. Constitutive interaction of TBK1 with OPTN and the ability of OPTN to bind to ubiquitin chains are essential for TBK1 recruitment and kinase activation on mitochondria. TBK1 in turn phosphorylates OPTN's UBAN domain at S473, thereby expanding the binding capacity of OPTN to diverse Ub chains. In combination with phosphorylation of S177 and S513, this posttranslational modification promotes recruitment and retention of OPTN/TBK1 on ubiquitinated, damaged mitochondria. Moreover, phosphorylation of OPTN on S473 enables binding to pS65 Ub chains and is also implicated in PINK1-driven and Parkin-independent mitophagy. Thus, TBK1-mediated phosphorylation of autophagy receptors creates a signal amplification loop operating in selective autophagy of damaged mitochondria.