Neuroimaging Features of Biotinidase Deficiency.

Biotinidase deficiency is an autosomal recessive condition caused by pathogenic variants in the BTD gene. Resultant deficiency of free biotin leads to impaired activity of the enzyme carboxylase and related neurologic, dermatologic, and ocular symptoms. Many of these are reversible on treatment, but early recognition and commencement of biotin supplementation are critical. This practice is especially important in countries where routine neonatal screening for biotinidase deficiency is not performed. In this report comprising 14 patients from multiple centers, we demonstrate the MR imaging patterns of this disorder at various age groups. Knowledge of these patterns in the appropriate clinical context will help guide early diagnosis of this treatable metabolic disorder.

Moderate Constraint Facilitates Association and Force-Dependent Dissociation of HA-CD44 Complex.

Binding of cell surface glycoprotein CD44 to hyaluronic acid (HA) is a key event for mediating cell adhesion, motility, metastasis, inflammatory responses and tumor development, but the regulation mechanism and its molecular basis under diverse mechanical constraints remain unclear. We herein investigated interaction of CD44 HABD (HA binding site domain) to HA through free and steered molecular dynamics (MD) simulations as well as atomic force microscope (AFM) measurement using different constraints on HA. The middle, two ends or both of the constrained HA chains were fixed for MD simulations, while one and two biotin-avidin linkage or physical absorption were used to immobilize HA on substrates for AFM experiments, to model HA chains with low, moderate and high HA flexibilities, respectively. We found that binding of CD44 to moderate fixed HA was possessed of a better thermo-stability, a lower mechanical strength and a higher dissociation probability, while higher adhesive frequency, smaller rupture force and shorter lifetime were assigned to CD44 on the two biotin-immobilized HA rather than one biotin-immobilized or physically absorbed HA on substrates, suggesting a moderate HA flexibility requirement in favor of association and force-induced dissociation of CD44-HA complex. Tensile-induced convex conformation of HA chain was responsible for reduction of complex mechano-stability and did inversely a shrunken CD44 HABD under stretching; transition from catch bond to slip bond governed CD44-HA interaction. This study uncovered the regulation mechanism and its molecular basis for CD44-HA affinity under diverse mechano-microenvironments and provided a new insight into CD44-HA interaction-mediated cell inflammatory responses and tumor development.

The opportunistic pathogen Pseudomonas aeruginosa exploits bacterial biotin synthesis pathway to benefit its infectivity.

Pseudomonas aeruginosa is an opportunistic pathogen that predominantly causes nosocomial and community-acquired lung infections. As a member of ESKAPE pathogens, carbapenem-resistant P. aeruginosa (CRPA) compromises the limited therapeutic options, raising an urgent demand for the development of lead compounds against previously-unrecognized drug targets. Biotin is an important cofactor, of which the de novo synthesis is an attractive antimicrobial target in certain recalcitrant infections. Here we report genetic and biochemical definition of P. aeruginosa BioH (PA0502) that functions as a gatekeeper enzyme allowing the product pimeloyl-ACP to exit from fatty acid synthesis cycle and to enter the late stage of biotin synthesis pathway. In relative to Escherichia coli, P. aeruginosa physiologically requires 3-fold higher level of cytosolic biotin, which can be attributed to the occurrence of multiple biotinylated enzymes. The BioH protein enables the in vitro reconstitution of biotin synthesis. The repertoire of biotin abundance is assigned to different mouse tissues and/or organ contents, and the plasma biotin level of mouse is around 6-fold higher than that of human. Removal of bioH renders P. aeruginosa biotin auxotrophic and impairs its intra-phagosome persistence. Based on a model of CD-1 mice mimicking the human environment, lung challenge combined with systemic infection suggested that BioH is necessary for the full virulence of P. aeruginosa. As expected, the biotin synthesis inhibitor MAC13772 is capable of dampening the viability of CRPA. Notably, MAC13772 interferes the production of pyocyanin, an important virulence factor of P. aeruginosa. Our data expands our understanding of P. aeruginosa biotin synthesis relevant to bacterial infectivity. In particular, this study represents the first example of an extracellular pathogen P. aeruginosa that exploits biotin cofactor as a fitness determinant, raising the possibility of biotin synthesis as an anti-CRPA target.

A toolbox for systematic discovery of stable and transient protein interactors in baker's yeast.

Identification of both stable and transient interactions is essential for understanding protein function and regulation. While assessing stable interactions is more straightforward, capturing transient ones is challenging. In recent years, sophisticated tools have emerged to improve transient interactor discovery, with many harnessing the power of evolved biotin ligases for proximity labelling. However, biotinylation-based methods have lagged behind in the model eukaryote, Saccharomyces cerevisiae, possibly due to the presence of several abundant, endogenously biotinylated proteins. In this study, we optimised robust biotin-ligation methodologies in yeast and increased their sensitivity by creating a bespoke technique for downregulating endogenous biotinylation, which we term ABOLISH (Auxin-induced BiOtin LIgase diminiSHing). We used the endoplasmic reticulum insertase complex (EMC) to demonstrate our approaches and uncover new substrates. To make these tools available for systematic probing of both stable and transient interactions, we generated five full-genome collections of strains in which every yeast protein is tagged with each of the tested biotinylation machineries, some on the background of the ABOLISH system. This comprehensive toolkit enables functional interactomics of the entire yeast proteome.

Discovery, structure, and function of filamentous 3-methylcrotonyl-CoA carboxylase.

3-methylcrotonyl-CoA carboxylase (MCC) is a biotin-dependent mitochondrial enzyme necessary for leucine catabolism in most organisms. While the crystal structure of recombinant bacterial MCC has been characterized, the structure and potential polymerization of native MCC remain elusive. Here, we discovered that native MCC from Leishmania tarentolae (LtMCC) forms filaments, and determined the structures of different filament regions at 3.4, 3.9, and 7.3 Å resolution using cryoEM. α6ß6 LtMCCs assemble in a twisted-stacks architecture, manifesting as supramolecular rods up to 400 nm. Filamentous LtMCCs bind biotin non-covalently and lack coenzyme A. Filaments elongate by stacking α6ß6 LtMCCs onto the exterior α-trimer of the terminal LtMCC. This stacking immobilizes the biotin carboxylase domains, sequestering the enzyme in an inactive state. Our results support a new model for LtMCC catalysis, termed the dual-swinging-domains model, and cast new light on the function of polymerization in the carboxylase superfamily and beyond.

Optimizing interpretation of survival studies of fresh and aged transfused biotin-labeled RBCs.

BACKGROUND: Ex vivo labeling with 51 chromium represents the standard method to determine red blood cell (RBC) survival after transfusion. Limitations and safety concerns spurred the development of alternative methods, including biotinylated red blood cells (BioRBC). STUDY DESIGN AND METHODS: Autologous units of whole blood were divided equally into two bags and stored under standard blood bank conditions at 2 to 6°C (N = 4 healthy adult volunteers). One bag was biotinylated (15 µg/ml) on storage days 5 to 7 (fresh) and the other was biotinylated (3 µg/ml) on days 35 to 42 (aged). The proportion of circulating BioRBC was measured serially, and cell-surface biotin was quantified with reference to molecules of equivalent soluble fluorochrome. Clearance kinetics were modeled by RBC age distribution at infusion (Gaussian vs. uniform) and decay over time (constant vs. exponential). RESULTS: Data were consistent with biphasic exponential clearance of cells of uniform age. Our best estimate of BioRBC clearance (half-life [T1/2 ]) was 49.7 ± 1.2 days initially, followed by more rapid clearance 82 days after transfusion (T1/2  = 15.6 ± 0.6 days). As BioRBC aged in vivo, molecules of equivalent soluble fluorochrome declined with a T1/2 of 122 ± 9 days, suggesting gradual biotin cleavage. There were no significant differences between the clearance of fresh and aged BioRBC. CONCLUSION: Similar clearance kinetics of fresh and aged BioRBC may be due to the extensive washing required during biotinylation. Survival kinetics consistent with cells with uniform rather than Gaussian or other non-uniform age distributions suggest that washing, and potentially RBC culling, may extend the storage life of RBC products.

Huntington disease oligodendrocyte maturation deficits revealed by single-nucleus RNAseq are rescued by thiamine-biotin supplementation.

The complexity of affected brain regions and cell types is a challenge for Huntington's disease (HD) treatment. Here we use single nucleus RNA sequencing to investigate molecular pathology in the cortex and striatum from R6/2 mice and human HD post-mortem tissue. We identify cell type-specific and -agnostic signatures suggesting oligodendrocytes (OLs) and oligodendrocyte precursors (OPCs) are arrested in intermediate maturation states. OL-lineage regulators OLIG1 and OLIG2 are negatively correlated with CAG length in human OPCs, and ATACseq analysis of HD mouse NeuN-negative cells shows decreased accessibility regulated by OL maturation genes. The data implicates glucose and lipid metabolism in abnormal cell maturation and identify PRKCE and Thiamine Pyrophosphokinase 1 (TPK1) as central genes. Thiamine/biotin treatment of R6/1 HD mice to compensate for TPK1 dysregulation restores OL maturation and rescues neuronal pathology. Our insights into HD OL pathology spans multiple brain regions and link OL maturation deficits to abnormal thiamine metabolism.

Coordinate-Dependent Drift-Diffusion Reveals the Kinetic Intermediate Traps of Top7-Based Proteins.

The computer-designed Top7 served as a scaffold to produce immunoreactive proteins by grafting of the 2F5 HIV-1 antibody epitope (Top7-2F5) followed by biotinylation (Top7-2F5-biotin). The resulting nonimmunoglobulin affinity proteins were effective in inducing and detecting the HIV-1 antibody. However, the grafted Top7-2F5 design led to protein aggregation, as opposed to the soluble biotinylated Top7-2F5-biotin. The structure-based model predicted that the thermodynamic cooperativity of Top7 increases after grafting and biotin-labeling, reducing their intermediate state populations. In this work, the folding kinetic traps that might contribute to the aggregation propensity are investigated by the diffusion theory. Since the engineered proteins have similar sequence and structural homology, they served as protein models to study the kinetic intermediate traps that were uncovered by characterizing the position-dependent drift-velocity (v(Q)) and the diffusion (D(Q)) coefficients. These coordinate-dependent coefficients were taken into account to obtain the folding and transition path times over the free energy transition states containing the intermediate kinetic traps. This analysis may be useful to predict the aggregated kinetic traps of scaffold-epitope proteins that might compose novel diagnostic and therapeutic platforms.

[Role of brain and muscle ARNT-like protein 1 in the rat periodontitis-induced liver injury model].

Objective: Brain and muscle ARNT-like protein 1 (BMAL1) is a core component of hepatocyte molecular clock and plays an important role in the regulation of other related rhythmic genes in the body through a transcriptional-translational feedback loop in molecular circadian oscillations. Therefore, the aim of this study was to investigate the role of BMAL1 in the rat periodontitis-induced liver injury. Methods: Twelve male Wistar rats were divided into the control group and the periodontitis group according to the random number table method. The rats in the control group were untreated. The periodontitis models were established by ligating the necks of the bilateral maxillary first molars in the periodontitis group rats. After 8 weeks, periodontal clinical indexes of rats in both groups were examined and executed. Micro-CT scans of the maxilla were performed and levels of the alveolar bone resorption were analyzed. Pathological changes in periodontal and liver tissue of rats in two groups were detected by HE and oil red O staining. Biochemical kits were used to detect glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), total cholesterol (TC) and triglycerides (TG) in serum. The gene and protein expression levels of BMAL1, nuclear factor kappa-B (NF-κB) and tumor necrosis factor-α (TNF-α) in liver tissue were measured by real time fluorescent quantitative-PCR (qRT-PCR), immunohistochemistry (IHC) and Western blotting (WB) assays. Apoptosis was detected in liver tissues by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) kit staining. Results: The results of HE staining of maxillary first molars and micro-CT results of maxillary bones showed that alveolar bone resorption was significant in the periodontitis group of rats. The liver histopathology results showed infiltrated inflammatory cells in the liver tissue, disorganized liver cords and a large number of lipid droplets formed in the hepatocytes of the periodontitis group compared with the control group. The results of serum biochemical assay showed that the levels of GOT [(62.77±2.59) U/L], GPT [(47.54±1.04) U/L], TC [(3.19±0.23) mmol/L] and TG [(1.11±0.09) mmol/L] in the serum of rats with periodontitis were significantly higher than that in the control group respectively [GOT: (38.66±2.47) U/L, GPT: (31.48±1.57) U/L, TC: (1.60±0.05) mmol/L and TG: (0.61±0.09) mmol/L](P=0.003, P=0.001, P=0.002, P=0.038). qRT-PCR results showed that the mRNA expression level of BMAL1 was significantly decreased in liver tissue of the periodontitis group [(0.60±0.04)%] compared to the control group [(1.01±0.07)%] (t=4.80, P=0.009), while the mRNA expression levels of NF-κB and TNF-α [(1.62±0.12)%, (2.69±0.16)%] were significantly increased compared to the control group [(1.00±0.03)%, (1.03±0.16)%] (P=0.008, P=0.002); IHC results showed that the protein expression level of BMAL1 in liver tissue of the periodontitis group (averaged optical density, AOD) (11.58±2.15) was down-regulated compared to the control group (AOD) (22.66±1.67) (P=0.015), while NF-κB and TNF-α (AOD) (31.77±2.69, 24.31±2.32) were up-regulated compared to the control group (AOD) (19.40±1.82, 11.92±0.94) (P=0.019, P=0.008). WB results showed that the protein expression level of BMAL1 in liver tissue was down-regulated in the periodontitis group [(0.63±0.10)%] compared to the control group [(1.00±0.06)%] (t=3.19, P=0.033), while NF-κB and TNF-α [(1.61±0.12)%, (2.82±0.23)%] were up-regulated compared to the control group [(1.00±0.12)%, (1.00±0.11)%] (P=0.022, P=0.002). TUNEL staining showed increased apoptotic cells in the liver tissue of the periodontitis group of rats compared to the control group. Conclusions: Periodontitis may induce liver injury by down-regulating the BMAL1 expression levels in liver tissue, which in turn activates NF-κB signaling molecules, leading to the elevated levels of inflammation and apoptosis in rat liver.

Activity-Induced Cortical Glutamatergic Neuron Nascent Proteins.

Neuronal activity initiates signaling cascades that culminate in diverse outcomes including structural and functional neuronal plasticity, and metabolic changes. While studies have revealed activity-dependent neuronal cell type-specific transcriptional changes, unbiased quantitative analysis of cell-specific activity-induced dynamics in newly synthesized proteins (NSPs) synthesis in vivo has been complicated by cellular heterogeneity and a relatively low abundance of NSPs within the proteome in the brain. Here we combined targeted expression of mutant MetRS (methionine tRNA synthetase) in genetically defined cortical glutamatergic neurons with tight temporal control of treatment with the noncanonical amino acid, azidonorleucine, to biotinylate NSPs within a short period after pharmacologically induced seizure in male and female mice. By purifying peptides tagged with heavy or light biotin-alkynes and using direct tandem mass spectrometry detection of biotinylated peptides, we quantified activity-induced changes in cortical glutamatergic neuron NSPs. Seizure triggered significant changes in ∼300 NSPs, 33% of which were decreased by seizure. Proteins mediating excitatory and inhibitory synaptic plasticity, including SynGAP1, Pak3, GEPH1, Copine-6, and collybistin, and DNA and chromatin remodeling proteins, including Rad21, Smarca2, and Ddb1, are differentially synthesized in response to activity. Proteins likely to play homeostatic roles in response to activity, such as regulators of proteastasis, intracellular ion control, and cytoskeleton remodeling proteins, are activity induced. Conversely, seizure decreased newly synthetized NCAM, among others, suggesting that seizure induced degradation. Overall, we identified quantitative changes in the activity-induced nascent proteome from genetically defined cortical glutamatergic neurons as a strategy to discover downstream mediators of neuronal plasticity and generate hypotheses regarding their function.SIGNIFICANCE STATEMENT Activity-induced neuronal and synaptic plasticity are mediated by changes in the protein landscape, including changes in the activity-induced newly synthesized proteins; however, identifying neuronal cell type-specific nascent proteome dynamics in the intact brain has been technically challenging. We conducted an unbiased proteomic screen from which we identified significant activity-induced changes in ∼300 newly synthesized proteins in genetically defined cortical glutamatergic neurons within 20 h after pharmacologically induced seizure. Bioinformatic analysis of the dynamic nascent proteome indicates that the newly synthesized proteins play diverse roles in excitatory and inhibitory synaptic plasticity, chromatin remodeling, homeostatic mechanisms, and proteasomal and metabolic functions, extending our understanding of the diversity of plasticity mechanisms.

Genome-wide identification of biotin carboxyl carrier subunits of acetyl-CoA carboxylase in Brassica and their role in stress tolerance in oilseed Brassica napus.

BACKGROUND: Biotin carboxyl carrier protein (BCCP) is a subunit of Acetyl CoA-carboxylase (ACCase) which catalyzes the conversion of acetyl-CoA to malonyl-CoA in a committed step during the de novo biosynthesis of fatty acids. Lipids, lipid metabolites, lipid-metabolizing and -modifying enzymes are known to play a role in biotic and abiotic stress tolerance in plants. In this regard, an understanding of the Brassica napus BCCP genes will aid in the improvement of biotic and abiotic stress tolerance in canola. RESULTS: In this study, we identified 43 BCCP genes in five Brassica species based on published genome data. Among them, Brassica rapa, Brassica oleracea, Brassica nigra, Brassica napus and Brassica juncea had six, seven, seven, 10 and 13 BCCP homologs, respectively. Phylogenetic analysis categorized them into five classes, each with unique conserved domains. The promoter regions of all BCCP genes contained stress-related cis-acting elements as determined by cis-element analysis. We identified four and three duplicated gene pairs (segmental) in B. napus and B. juncea respectively, indicating the role of segmental duplication in the expansion of this gene family. The Ka/Ks ratios of orthologous gene pairs between Arabidopsis thaliana and five Brassica species were mostly less than 1.0, implying that purifying selection, i.e., selective removal of deleterious alleles, played a role during the evolution of Brassica genomes. Analysis of 10 BnaBCCP genes using qRT-PCR showed a different pattern of expression because of exposure of the plants to biotic stresses, such as clubroot and sclerotinia diseases, and abiotic stresses such as drought, low temperature and salinity stresses. CONCLUSIONS: The identification and functional analysis of the Brassica BCCPs demonstrated that some of these genes might play important roles in biotic and abiotic stress responses. Results from this study could lay the foundation for a better understanding of these genes for the improvement of Brassica crops for stress tolerance.

Identification of MPK4 kinase interactome using TurboID proximity labeling proteomics in Arabidopsis thaliana.

TurboID is a new and efficient proximity labeling system that was first developed in living mammalian cells. TurboID is a modified bacterial biotin ligase that can be fused to a bait protein, which can then modify proximal interacting proteins with biotin. Prey proteins subsequently labeled with biotin tags will be pulled down with streptavidin-coated beads and identified by mass spectrometry-based proteomics. TurboID has been recently applied to living plant cells and provided promising results in identification of interacting proteins. Mitogen-activated protein kinase 4 (MPK4) is important for plant growth, development, and defense; however, the molecular mechanisms underlying the range of MPK4 functions are not completely known. Here we use modern proteomics together with the TurboID in a proof-of-concept study to profile the MPK4 interactome and uncover the functions of MPK4 in plant signaling cascades.

Nitric oxide alleviates salt stress through protein S-nitrosylation and transcriptional regulation in tomato seedlings.

MAIN CONCLUSION: NO enhances the resistance of tomato seedlings to salt stress through protein S-nitrosylation and transcriptional regulation, which involves the regulation of MAPK signaling and carbohydrate metabolism. Nitric oxide (NO) regulates various physiological and biochemical processes and stress responses in plants. We found that S-nitrosoglutathione (GSNO) treatment significantly promoted the growth of tomato seedling under NaCl stress, indicating that NO plays a positive role in salt stress resistance. Moreover, GSNO pretreatment resulted in an increase of endogenous NO level, S-nitrosothiol (SNO) content, S-nitrosoglutathione reductase (GSNOR) activity and GSNOR expression under salt stress, implicating that S-nitrosylation might be involved in NO-alleviating salt stress. To further explore whether S-nitrosylation is a key molecular mechanism of NO-alleviating salt stress, the biotin-switch technique and liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) were conducted. A total of 1054 putative S-nitrosylated proteins have been identified, which were mainly enriched in chloroplast, cytoplasm and mitochondrion. Among them, 15 and 22 S-nitrosylated proteins were involved in mitogen-activated protein kinase (MAPK) signal transduction and carbohydrate metabolism, respectively. In MAPK signaling, various S-nitrosylated proteins, SAM1, SAM3, SAM, PP2C and SnRK, were down-regulated and MAPK, MAPKK and MAPKK5 were up-regulated at the transcriptional level by GSNO treatment under salt stress compared to NaCl treatment alone. The GSNO pretreatment could reduce ethylene production and ABA content under NaCl stress. In addition, the activities of enzyme identified in carbohydrate metabolism, their expression at the transcriptional level and the metabolite content were up-regulated by GSNO supplication under salt stress, resulting in the activation of glycolysis and tricarboxylic acid cycle (TCA) cycles. Thus, these results demonstrated that NO might beneficially regulate MAPK signaling at transcriptional levels and activate carbohydrate metabolism at the post-translational and transcriptional level, protecting seedlings from energy deficiency and salinity, thereby alleviating salt stress-induced damage in tomato seedlings. It provides initial insights into the regulatory mechanisms of NO in response to salt stress.

LncRNA-SUSAJ1 Activates the ER Stress Pathway Inhibiting JEV Proliferation by Promoting PK15 Cells Apoptosis.

BACKGROUND: Epidemic encephalitis B is a common zoonosis that threatens both pigs and humans. Effective prevention and control of epidemic encephalitis B is difficult. The cellular defence mechanism is closely related to the body's resistance to viral invasion. Long non-coding RNAs (lncRNAs) are involved in regulating various cellular activities. We previously found that lncRNA-SUSAJ1 could inhibit the proliferation of Japanese encephalitis virus (JEV). However, the mechanism underlying this suppression remains unclear. METHODS: We performed Western blotting and quantitative reverse-transcription polymerase chain reaction (RT-qPCR) analyses, as well as mitochondrial membrane potential, flow cytometry, terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL), RNA pull-down, and RNA immunoprecipitation assays. RESULTS: JC-1 cationic dye staining showed that lncRNA-SUSAJ1 promoted the depolarisation of mitochondrial membrane potential; H2DCFDA probe staining showed that lncRNA-SUSAJ1 enhanced the level of reactive oxygen species in PK15 porcine kidney cells. qRT-PCR and Western blotting revealed the expression levels of associated mRNAs and proteins, and the TUNEL and flow cytometry assays detected cell apoptosis. Their results showed that lncRNA-SUSAJ1 promoted the expression of pro-apoptotic genes and inhibited the expression of anti-apoptotic genes. RNA pull-down experiments using biotin-labelled lncRNA-SUSAJ1 showed colocalisation between lncRNA-SUSAJ1 and the 70 kDa heat shock protein (Hsp70). lncRNA-SUSAJ1 also activated unfolded protein response-related pathways, regulated protein degradation, and promoted apoptosis via the endoplasmic reticulum stress response, thereby inhibiting viral replication. CONCLUSIONS: The findings of this study provide insight into the specific molecular mechanism of lncRNA-SUSAJ1 resistance to viral proliferation by promoting cell apoptosis, clarify the antiviral effect of lncRNA-SUSAJ1 on JEV.

Polo-Like Kinase 2 Plays an Essential Role in Cytoprotection against MG132-Induced Proteasome Inhibition via Phosphorylation of Serine 19 in HSPB5.

Protein homeostasis, including protein folding, refolding, and degradation, is thought to decline with aging. HSPB5 (also known as αB-crystallin) prevents target protein aggregation as a molecular chaperone and exhibits a cytoprotective function against various cell stresses. To elucidate the effect of HSPB5 on endoplasmic reticulum (ER) stress, we searched for novel binding proteins of HSPB5 using the proximity-dependent biotin labeling method. Proteins presumed to interact with HSPB5 in cells treated with the proteasome inhibitor MG132 were identified by a reversible biotin-binding capacity method combining tamavidin2-REV magnetic beads and mass spectrometry. We discovered a new binding protein for HSPB5, polo-like kinase 2 (PLK2), which is an apoptosis-related enzyme. The expression of PLK2 was upregulated by MG132 treatment, and it was co-localized with HSPB5 near the ER in L6 muscle cells. Inhibition of PLK2 decreased ER stress-induced phosphorylation of serine 19 in HSPB5 and increased apoptosis by activation of caspase 3 under ER stress. Overexpression of HSPB5 (WT) suppressed the ER stress-induced caspase 3 activity, but this was not observed with phospho-deficient HSPB5 (3A) mutants. These results clarify the role of HSPB5 phosphorylation during ER stress and suggest that the PLK2/HSPB5 pathway plays an essential role in cytoprotection against proteasome inhibition-induced ER stress.

Two novel platelet biotinylation methods and their impact on stored platelet concentrates in a blood bank environment.

BACKGROUND: Storage of platelet concentrates (PCs) has an impact on platelet quality and possibly affects their functions after transfusion. The influence of processing and storage conditions of PCs on their in vivo function upon transfusion is unknown. One option for investigating this question is to implement an ex vivo labeling of human platelets, to analyze them after transfusion into heathy volunteers and/or patients. In this study, we developed two labeling methods employing biotin. METHODS: Two methods of biotinylation were compared to a control (standard PC). The "Bio-Wash" process used washing steps to label all platelets within the PC; for the other method, "Bio-Direct," one fifth of the PC were directly labeled without washing steps. The control and the two biotinylated PCs were analyzed over 7 days of storage. Labeling efficiency, platelet counts, phenotypes, and functions, along with time and costs, were evaluated to select the best process. RESULTS: Both methods achieved a stable labeling through the storage, with similar platelet counts and metabolism in comparison to control PCs. Bio-Wash showed higher activation phenotype and lower aggregation response in comparison to the Bio-Direct method. The Bio-Direct was performed within 1.5 h versus 3 h for the Bio-Wash. However, the Bio-Direct required 12 mg of biotin instead of 8 mg for the other process. CONCLUSION: We set up two methods of biotinylation that can be easily implemented in a blood bank environment. The Bio-Direct process was preferred to the Bio-Wash because of its similarity, from a functional and phenotypic point of view, with standard PCs.

A microfluidics-based method for isolation and visualization of cells based on receptor-ligand interactions.

Receptor-ligand binding has been analyzed at the protein level using isothermal titration calorimetry and surface plasmon resonance and at the cellular level using interaction-associated downstream gene induction/suppression. However, no currently available technique can characterize this interaction directly through visualization. In addition, all available assays require a large pool of cells; no assay capable of analyzing receptor-ligand interactions at the single-cell level is publicly available. Here, we describe a new microfluidic chip-based technique for analyzing and visualizing these interactions at the single-cell level. First, a protein is immobilized on a glass slide and a low-flow-rate pump is used to isolate cells that express receptors that bind to the immobilized ligand. Specifically, we demonstrate the efficacy of this technique by immobilizing biotin-conjugated FGL2 on an avidin-coated slide chip and passing a mixture of GFP-labeled wild-type T cells and RFP-labeled FcγRIIB-knockout T cells through the chip. Using automated scanning and counting, we found a large number of GFP+ T cells with binding activity but significantly fewer RFP+ FcγRIIB-knockout T cells. We further isolated T cells expressing a membrane-anchored, tumor-targeted IL-12 based on the receptor's affinity to vimentin to confirm the versatility of our technique. This protocol allows researchers to isolate receptor-expressing cells in about 4 hours for further downstream processing.

Y98 Mutation Leads to the Loss of RsfS Anti-Association Activity in Staphylococcus aureus.

Ribosomal silencing factor S (RsfS) is a conserved protein that plays a role in the mechanisms of ribosome shutdown and cell survival during starvation. Recent studies demonstrated the involvement of RsfS in the biogenesis of the large ribosomal subunit. RsfS binds to the uL14 ribosomal protein on the large ribosomal subunit and prevents its association with the small subunit. Here, we estimated the contribution of RsfS amino acid side chains at the interface between RsfS and uL14 to RsfS anti-association function in Staphylococcus aureus through in vitro experiments: centrifugation in sucrose gradient profiles and an S. aureus cell-free system assay. The detected critical Y98 amino acid on the RsfS surface might become a new potential target for pharmacological drug development and treatment of S. aureus infections.

BR-Mediated Protein S-Nitrosylation Alleviated Low-Temperature Stress in Mini Chinese Cabbage (Brassica rapa ssp. pekinensis).

Brassinosteroids (BRs), a novel plant hormone, are widely involved in plant growth and stress response processes. Nitric oxide (NO), as an important gas signaling molecule, can regulate target protein activity, subcellular localization and function in response to various stresses through post-translational S-nitrosylation modifications. However, the relationship between BR and NO in alleviating low-temperature stress of mini Chinese cabbage remains unclear. The hydroponic experiment combined with the pharmacological and molecular biological method was conducted to study the alleviating mechanism of BR at low temperature in mini Chinese cabbage. The results showed that low temperature inhibited the growth of mini Chinese cabbage seedlings, as evidenced by dwarf plants and yellow leaves. Treatment with 0.05 mg/L BR and 50 µM NO donor S-nitrosoglutathione (GSNO) significantly increased the leaf area, stem diameter, chlorophyll content, dry and fresh weight and proline content. Meanwhile, the malondialdehyde (MDA) content in 0.05 mg/L BR- and 50 µM GSNO-treated leaves were significantly lower than those in other treated leaves under low-temperature conditions. In addition, BR and GSNO applications induced an increase in NO and S-nitrosothiol (SNO) levels in vivo under low-temperature stress. Similarly, spraying BR after the elimination of NO also increased the level of S-nitrosylation in vivo, while spraying GSNO after inhibiting BR biosynthesis decreased the level of NO and SNO in vivo. In contrast, the S-nitrosoglutathione reductase (BrGSNOR) relative expression level and GSNOR enzyme activity were downregulated and inhibited by BR treatment, GSNO treatment and spraying BR after NO clearance, while the relative expression level of BrGSNOR was upregulated and GSNOR enzyme activity was also increased when spraying GSNO after inhibiting BR synthesis. Meanwhile, the biotin switch assay showed that exogenous BR increased the level of total nitrosylated protein in vivo under low-temperature stress. These results suggested that BR might act as an upstream signal of NO, induced the increase of NO content in vivo and then induced the protein S-nitrosylation modification to alleviate the damage of mini Chinese cabbage seedlings under low-temperature stress.

DNA-functionalized colloidal crystals for macromolecular encapsulation.

Novel DNA-based structures with the ability to encapsulate nanoscale molecules, such as proteins, can be applied to a wide range of areas, including reaction fields and micro/nano drug carriers. DNA-functionalized nanoparticle (DNA-NP) colloidal crystals have emerged as a new class of programmable DNA-based structures harboring metal nanoparticles with improved mechanical properties. The encapsulation of guest molecules into empty spaces in lattice structures is theoretically possible. However, due to the lack of a strategy for versatile encapsulation of guest molecules, the feasibility of nanoscale encapsulation by DNA-NP crystals is unclear. In this study, we developed DNA-functionalized gold nanoparticle (DNA-AuNP) crystals with tunable interparticle spacing for molecular encapsulation. We demonstrated that the modification of DNA-AuNP crystals with functional moieties, that is, biotin molecules, was effective in retaining molecules in the crystals. The crystallinities before and after encapsulation of the molecules were confirmed using small-angle X-ray scattering. We also succeeded in encapsulating CRISPR/Cas9 ribonucleoproteins into DNA-AuNP crystals by harnessing their affinity for target molecules. These findings demonstrated the potential use of metal-DNA hybrid crystals as carriers for direct protein delivery via biolistic bombardment. Thus, this study provides an attractive strategy for creating a new class of DNA-based structures for macromolecular encapsulation, and an alternative research direction toward colloidal crystal engineering using DNA.