In vitro cleavage of tumor necrosis factor α (TNFα) by Signal-Peptide-Peptidase-like 2b (SPPL2b) resembles mechanistic principles observed in the cellular context.

Members of the Signal Peptide-Peptidase (SPP) and Signal Peptide-Peptidase-like (SPPL) family are intramembrane aspartyl-proteases like their well-studied homologs, the presenilins, which comprise the catalytically active subunit within the γ-secretase complex. The lack of in vitro cleavage assays for SPPL proteases limited their biochemical characterization as well as substrate identification and validation. So far, SPPL proteases have been analyzed exclusively in intact cells or membranes, restricting mechanistic analysis to co-expression of enzyme and substrate variants colocalizing in the same subcellular compartments. We describe the details of developing an in vitro cleavage assay for SPPL2b and its model substrate TNFα and analyzed the influence of phospholipids, detergent supplements, and cholesterol on the SPPL2b in vitro activity. SPPL2b in vitro activity resembles mechanistic principles that have been observed in a cellular context, such as cleavage sites and consecutive turnover of the TNFα transmembrane domain. The novel in vitro cleavage assay is functional with separately isolated protease and substrate and amenable to a high throughput plate-based readout overcoming previous limitations and providing the basis for studying enzyme kinetics, catalytic activity, substrate recognition, and the characteristics of small molecule inhibitors. As a proof of concept, we present the first biochemical in vitro characterization of the SPPL2a and SPPL2b specific small molecule inhibitor SPL-707.

Efficient production of isomaltulose using engineered Yarrowia lipolytica strain facilitated by non-yeast signal peptide-mediated cell surface display.

BACKGROUND: Isomaltulose is a 'generally recognized as safe' ingredient and is widely used in the food, pharmaceutical and chemical industries. The exploration and development of efficient technologies is essential for enhancing isomaltulose yield. RESULTS: In the present study, a simple and efficient surface display platform mediated by a non-yeast signal peptide was developed in Yarrowia lipolytica and utilized to efficiently produce isomaltulose from sucrose. We discovered that the signal peptide SP1 of sucrose isomerase from Pantoea dispersa UQ68J (PdSI) could guide SIs anchoring to the cell surface of Y. lipolytica, demonstrating a novel and simple cell surface display strategy. Furthermore, the PdSI expression level was significantly increased through optimizing the promoters and multi-site integrating genes into chromosome. The final strain gained 451.7 g L-1 isomaltulose with a conversion rate of 90.3% and a space-time yield of 50.2 g L-1 h-1 . CONCLUSION: The present study provides an efficient way for manufacturing isomaltulose with a high space-time yield. This heterogenous signal peptide-mediated cell surface display strategy featured with small fusion tag (approximately 2.2 kDa of SP1), absence of enzyme leakage in fermentation broth and ample room for optimization, providing a convenient way to construct whole-cell biocatalysts to synthesize other products and broadening the array of molecular toolboxes accessible for engineering Y. lipolytica. © 2024 Society of Chemical Industry.

[Analysis of signal peptides and secreted proteins in the whole proteome of Pichia pastoris].

The signal peptide is a key factor that affects the efficiency of protein secretion in Pichia pastoris. Currently, the most used signal peptide is the α-mating factor (MFα) pre-pro leader from Saccharomyces cerevisiae. This exogenous signal peptide has been successfully utilized to express and secret many heterologous proteins. However, MFα is not suitable for the secretory expression of all heterologous proteins. Many typical signal peptides are present in the secretory proteins of P. pastoris, which provides more options besides MFα. Therefore, it is necessary to analyze and identify more efficient endogenous signal peptides that can guide the secretion of heterologous proteins in P. pastoris. In this study, we employed bioinformatics tools such as SignalP, TMHMM, Phobius, WoLF PSORT, and NetGPI to predict endogenous signal peptides from the entire proteome of P. pastoris GS115 (ATCC 20864). Moreover, we analyzed the distribution, length, amino acid composition, and conservation of these signal peptides. Additionally, we screened 69 secreted proteins and their signal peptides, and through secretome validation, we identified 10 endogenous signal peptides that have potential to be used for exogenous protein expression. The endogenous signal peptides obtained in this study may serve as new valuable tools for the expression and secretion of heterologous proteins in P. pastoris.

Comparison of the efficiency of the Sec and Tat secretory pathways in the secretion of recombinant neurturin protein using de novo designed signal peptides.

Since cytoplasmic expression of heterologous proteins with disulfide bonds leads to the formation of inclusion bodies in E. coli, periplasmic production is preferable. The N-terminal signal peptide attached to the secreted protein determines the type of secretory pathway through which the target protein is secreted; Sec, Tat, or SRP. The aim of this study was to design and compare two novel signal peptides for the secretion of recombinant neurturin (as a model) via the Sec and Tat pathways. For this purpose, we aligned the natural signal peptides from E. coli and Bacillus subtilis to identify the conserved amino acids and those with the highest repetition. The SignalP4.1 and TatP1.0 software were used to determine the secretion efficiency of the new signal peptides. The efficiency of new signal peptides was then evaluated and compared experimentally with two naturally used signal peptides. Quantitative analysis of Western blot bands showed that approximately 80% of the expressed neurturin was secreted into the periplasmic space by new signal peptides. Circular dichroism spectroscopy also confirmed the correct secondary structure of the secreted neurturin. In conclusion, these novel signal peptides can be used to secrete any other recombinant proteins to the periplasmic space of E. coli efficiently.

Signal Peptide Peptidase and PI4Kß1/2 play opposite roles in plant ER stress response and immunity.

The Arabidopsis pi4kß1,2 mutant is mutated in the phosphatidylinositol 4-kinase (PI4K) ß1 and PI4Kß2 enzymes which are involved in the biosynthesis of phosphatidylinositol 4-phosphate (PI4P), a minor membrane lipid with important signaling roles. pi4kß1,2 plants display autoimmunity and shorter roots. Though the pi4kß1,2 mutant has been extensively characterized, the source of its autoimmunity remains largely unknown. In this study, through a genetic suppressor screen, we identified multiple partial loss-of-function alleles of signal peptide peptidase (spp) that can suppress all the defects of pi4kß1,2. SPP is an intramembrane cleaving aspartic protease. Interestingly, pi4kß1,2 plants display enhanced ER stress response and mutations in SPP can suppress such phenotype. Furthermore, reduced ER stress responses were observed in the spp single mutants. Overall, our study reveals a previously unknown function of PI4Kß and SPP in ER stress and plant immunity.

Exploiting Systematic Engineering of the Expression Cassette as a Powerful Tool to Enhance Heterologous Gene Expression in Trichoderma reesei.

Many endeavors in expressing a heterologous gene in microbial hosts rely on simply placing the gene of interest between a selected pair of promoters and terminator. However, although the expression efficiency could be improved by engineering the host cell, how modifying the expression cassette itself systematically would affect heterologous gene expression remains largely unknown. As the promoter and terminator bear plentiful cis-elements, herein using the Aspergillus niger mannanase with high application value in animal feeds and the eukaryotic filamentous fungus workhorse Trichoderma reesei as a model gene/host, systematic engineering of an expression cassette was investigated to decipher the effect of its mutagenesis on heterologous gene expression. Modifying the promoter, signal peptide, the eukaryotic-specific Kozak sequence, and the 3'-UTR could stepwise improve extracellular mannanase production from 17 U/mL to an ultimate 471 U/mL, representing a 27.7-fold increase in expression. The strategies can be generally applied in improving the production of heterologous proteins in eukaryotic microbial hosts.

Highly efficient expression of Rasamsonia emersonii lipase in Pichia pastoris: characterization and gastrointestinal simulated digestion in vitro.

BACKGROUND: Acidic lipases with high catalytic activities under acidic conditions have important application values in the food, feed and pharmaceutical industries. However, the availability of acidic lipases is still the main obstacle to their industrial applications. Although a novel acidic lipase Rasamsonia emersonii (LIPR) was heterologously expressed in Escherichia coli, the expression level was unsatisfactory. RESULTS: To achieve the high-efficiency expression and secretion of LIPR in Pichia pastoris GS115, the combinatorial optimization strategy was adopted including gene codon preference, signal peptide, molecular chaperone co-expression and disruption of vacuolar sorting receptor VPS10. The activity of the combinatorial optimization engineered strain in a shake flask reached 1480 U mL-1 , which was 8.13 times greater than the P. pastoris GS115 parental strain. After high-density fermentation in a 5-L bioreactor, the highest enzyme activity reached as high as 11 820 U mL-1 . LIPR showed the highest activity at 40 °C and pH 4.0 in the presence of Ca2+ ion. LIPR exhibited strong tolerance to methanol, indicating its potential application in biodiesel biosynthesis. Moreover, the gastrointestinal digestion simulation results demonstrated that LIPR was tolerant to pepsin and trypsin, but its activity was inhibited by sodium taurodeoxycholate. CONCLUSION: This study provided an effective approach for the high expression of acidic lipase LIPR. LIPR was more appropriate for lipid digestion in the stomach than in intestine according to the gastrointestinal digestion simulation results. © 2024 Society of Chemical Industry.

Identification of novel tail-anchored membrane proteins integrated by the bacterial twin-arginine translocase.

The twin-arginine protein transport (Tat) system exports folded proteins across the cytoplasmic membranes of prokaryotes and the energy transducing-membranes of plant thylakoids and mitochondria. Proteins are targeted to the Tat machinery by N-terminal signal peptides with a conserved twin-arginine motif, and some substrates are exported as heterodimers where the signal peptide is present on one of the partner proteins. A subset of Tat substrates is found in the membrane. Tat-dependent membrane proteins usually have large globular domains and a single transmembrane helix present at the N- or C-terminus. Five Tat substrates that have C-terminal transmembrane helices have previously been characterized in the model bacterium Escherichia coli. Each of these is an iron-sulfur cluster-containing protein involved in electron transfer from hydrogen or formate. Here we have undertaken a bioinformatic search to identify further tail-anchored Tat substrates encoded in bacterial genomes. Our analysis has revealed additional tail-anchored iron-sulfur proteins associated in modules with either a b-type cytochrome or a quinol oxidase. We also identified further candidate tail-anchored Tat substrates, particularly among members of the actinobacterial phylum, that are not predicted to contain cofactors. Using reporter assays, we show experimentally that six of these have both N-terminal Tat signal peptides and C-terminal transmembrane helices. The newly identified proteins include a carboxypeptidase and a predicted protease, and four sortase substrates for which membrane integration is a prerequisite for covalent attachment to the cell wall.

A novel method for high level production of protein glutaminase by sfGFP tag in Bacillus subtilis.

Protein glutaminase (PG; EC 3.5.1.44) is a novel deamidase that helps to improve functional properties of food proteins. Currently, the highest activated PG enzyme activity was 26 U/mg when recombinantly expressed via the twin-arginine translocation (Tat) pathway in Corynebacterium glutamicum. In this study, superfolder green fluorescent protein (sfGFP) was used to replace traditional signal peptides to facilitate efficient heterologous expression and secretion of Propeptide-Protein glutaminase (PP) in Bacillus subtilis. The fusion protein, sfGFP-PP, was secreted from 12 h of fermentation and reached its highest extracellular expression at 28 h, with a secretion efficiency of about 93 %. Moreover, when fusing sfGFP with PP at the N-terminus, it significantly enhances PG expression up to 26 U/mL by approximately 2.2-fold compared to conventional signal-peptides- guided PP with 11.9 U/mL. Finally, the PG enzyme activity increased from 26 U/mL to 36.9 U/mL after promoter and RBS optimization. This strategy not only provides a new approach to increase PG production as well as extracellular secretion but also offers sfGFP as an effective N-terminal tag for increased secreted production of difficult-to-express proteins.

Loss of Preproinsulin Interaction with Signal Recognition Particle Activates Protein Quality Control, Decreasing mRNA Stability.

Many insulin gene variants alter the protein sequence and result in monogenic diabetes due to insulin insufficiency. However, the molecular mechanisms of various disease-causing mutations are unknown. Insulin is synthesized as preproinsulin containing a signal peptide (SP). SPs of secreted proteins are recognized by the signal recognition particle (SRP) or by another factor in a SRP-independent pathway. If preproinsulin uses SRP-dependent or independent pathways is still debatable. We demonstrate by the use of site-specific photocrosslinking that the SRP subunit, SRP54, interacts with the preproinsulin SP. Moreover, SRP54 depletion leads to the decrease of insulin mRNA and protein expression, supporting the involvement of the RAPP protein quality control in insulin biogenesis. RAPP regulates the quality of secretory proteins through degradation of their mRNA. We tested five disease-causing mutations in the preproinsulin SP on recognition by SRP and on their effects on mRNA and protein levels. We demonstrate that the effects of mutations are associated with their position in the SP and their severity. The data support diverse molecular mechanisms involved in the pathogenesis of these mutations. We show for the first time the involvement of the RAPP protein quality control pathway in insulin biogenesis that is implicated in the development of neonatal diabetes caused by the Leu13Arg mutation.

High-Level Extracellular Expression of Hyaluronate Lyase HylP in Bacillus subtilis for Hyaluronan Degradation.

Hyaluronate lyase (HA lyase) has potential in the industrial processing of hyaluronan. In this study, HylP, an HA lyase from Streptococcus pyogenes phage (SPB) was successfully expressed in Bacillus subtilis. To improve the extracellular enzyme activity of HylP in B. subtilis, signal peptide engineering systematic optimization was carried out, and cultured it from shake flasks and fermenters, followed by purification, characterization, and analysis of degradation products. The results showed that the replacement of the signal peptide increased the extracellular enzyme activity of HylP from 1.0 × 104 U/mL to 1.86 × 104 U/mL in the shake flask assay, and using a 20 L fermenter in a batch fermentation process, the extracellular enzyme activity achieved the level of 1.07 × 105 U/mL. HylP exhibited significant thermal and pH stability in the temperature range of 40 °C and pH range of 4-8, respectively. The enzyme showed optimum activity at 40 °C and pH 6, with significant activity in the presence of Na+, Mg2+, and Co2+ ions. Degradation analysis showed that HylP efficiently degraded hyaluronan as an endonuclease, releasing unsaturated disaccharides. These comprehensive findings underscore the substantial industrial potential of HylP for hyaluronan processing applications, offering valuable insights into enzyme characterization and optimization of expression for potential industrial utilization.

[Effect of signal peptide optimized by site-directed mutagenesis on the function of chimeric antigen receptor T cells].

Signal peptides (SP) are involved in regulating the secretion level and transmembrane translocation of chimeric antigen receptors (CAR), which is crucial for CAR-T cells. This study aimed to optimize the SP sequence by site-directed mutagenesis and investigate its impact on the killing function of CD19-CAR-T. Firstly, CAR vectors targeting CD19 containing wild-type SP (SP-wtY) or two mutant SP (SP-muK or SP-muR) were constructed using gene synthesis and molecular cloning techniques. The successfully constructed vector was packaged with lentivirus, and T cells were infected. The transfection efficiency of T cells was detected by flow cytometry, while the killing effect on target cells was assessed using the calcein release method. The secretion levels of cytokines interferon-γ (IFN-γ) and interferon-α (TNF-α) were measured using enzyme linked immunosorbent assay (ELISA). The results showed that successful construction of recombinant lentivirus plasmids with wild type and signal peptide mutation. After the transferring the lentivirus into T cells, the transfection efficiency of CD19-CAR carrying three signal peptides (SP-wtY, SP-muK, or SP-muR) were 33.9%, 35.5%, and 36.8%, respectively. Further killing assay showed that the tumor-killing effect of SP-muR cells was significantly higher than that of SP-muK and SP-wtY cells. When the ratio of effector to target was 10:1, the secretion levels of cytokines IFN-γ and TNF-α of CAR-T cells of the SP-muR group were significantly higher than those in SP-muK and SP-wtY groups. In summary, this study revealed that increasing the N-terminal positive charge of the signal peptide can improve the expression efficiency of CAR and promote the killing of CD19+ target cells. These findings provide a scientific basis the optimization and clinical application of CAR structure.

Novel Signal Peptides and Episomal Plasmid System for Enhanced Protein Secretion in Engineered Bacteroides Species.

The genus Bacteroides, a predominant group in the human gut microbiome, presents significant potential for microbiome engineering and the development of live biotherapeutics aimed at treating gut diseases. Despite its promising capabilities, tools for effectively engineering Bacteroides species have been limited. In our study, we have made a breakthrough by identifying novel signal peptides in Bacteroides thetaiotaomicron and Akkermansia muciniphila. These peptides facilitate efficient protein transport across cellular membranes in Bacteroides, a critical step for therapeutic applications. Additionally, we have developed an advanced episomal plasmid system. This system demonstrates superior protein secretion capabilities compared to traditional chromosomal integration plasmids, making it a vital tool for enhancing the delivery of therapeutic proteins in Bacteroides species. Initially, the stability of this episomal plasmid posed a challenge; however, we have overcome this by incorporating an essential gene-based selection system. This novel strategy not only ensures plasmid stability but also aligns with the growing need for antibiotic-free selection methods in clinical settings. Our work, therefore, not only provides a more robust secretion system for Bacteroides but also sets a new standard for the development of live biotherapeutics.

Complementary hydrophobic interaction of the redox enzyme maturation protein NarJ with the signal peptide of the respiratory nitrate reductase NarG.

In bacteria, NarJ plays an essential role as a redox enzyme maturation protein in the assembly of the nitrate reductase NarGHI by interacting with the N-terminal signal peptide of NarG to facilitate cofactor incorporation into NarG. The purpose of our research was to elucidate the exact mechanism of NarG signal peptide recognition by NarJ. We determined the structures of NarJ alone and in complex with the signal peptide of NarG via X-ray crystallography and verified the NarJ-NarG interaction through mutational, binding, and molecular dynamics simulation studies. NarJ adopts a curved α-helix bundle structure with a U-shaped hydrophobic groove on its concave side. This groove accommodates the signal peptide of NarG via a dual binding mode in which the left and right parts of the NarJ groove each interact with two consecutive hydrophobic residues from the N- and C-terminal regions of the NarG signal peptide, respectively, through shape and chemical complementarity. This binding is accompanied by unwinding of the helical structure of the NarG signal peptide and by stabilization of the NarG-binding loop of NarJ. We conclude that NarJ recognizes the NarG signal peptide through a complementary hydrophobic interaction mechanism that mediates a structural rearrangement.

High prevalence of renal salt wasting induced by haptoglobin-related protein without signal peptide is linked to new syndrome of salt wasting in Alzheimer disease.

The subject of hyponatremia is undergoing significant changes after developing a more pathophysiologic approach that is superior to the ineffective volume approach and can more effectively identify the different causes of hyponatremia. This new approach identified cerebral salt wasting (CSW) in 24 (38%) of 62 hyponatremic patients from the medical wards of the hospital with 21 showing no evidence of cerebral disease to support our proposal to change CSW to renal salt wasting (RSW). RSW had to be differentiated from the syndrome of inappropriate secretion of antidiuretic hormone (SIADH) because of diametrically opposite therapeutic goals of water-restricting water-logged patients with SIADH or administering salt water to volume-depleted patients with RSW. Both syndromes present with identical clinical parameters that require a difficult protocol to make such a differentiation possible. We describe rat clearance studies demonstrating natriuretic activity in the plasma of patients with neurosurgical and Alzheimer diseases (AD) and eventually identify the protein as haptoglobin-related protein without signal peptide, which can serve as a biomarker to simplify diagnosis of RSW and delivery of the proper management to improve clinical outcomes. We also discuss the introduction of a new syndrome of RSW in AD and its implications. The high prevalence of RSW and identification of the natriuretic factor have created debates over the existence of RSW with none questioning or addressing the pathophysiologic data that identified patients with RSW. We also discuss the potentially large group of patients with RSW who are normonatremic.

The role of SPP/SPPL intramembrane proteases in membrane protein homeostasis.

Signal peptide peptidase (SPP) and the four SPP-like proteases SPPL2a, SPPL2b, SPPL2c and SPPL3 constitute a family of aspartyl intramembrane proteases with homology to presenilins. The different members reside in distinct cellular localisations within the secretory pathway and the endo-lysosomal system. Despite individual cleavage characteristics, they all cleave single-span transmembrane proteins with a type II orientation exhibiting a cytosolic N-terminus. Though the identification of substrates is not complete, SPP/SPPL-mediated proteolysis appears to be rather selective. Therefore, according to our current understanding cleavage by SPP/SPPL proteases rather seems to serve a regulatory function than being a bulk proteolytic pathway. In the present review, we will summarise our state of knowledge on SPP/SPPL proteases and in particular highlight recently identified substrates and the functional and/or (patho)-physiological implications of these cleavage events. Based on this, we aim to provide an overview of the current open questions in the field. These are connected to the regulation of these proteases at the cellular level but also in context of disease and patho-physiological processes. Furthermore, the interplay with other proteostatic systems capable of degrading membrane proteins is beginning to emerge.

Hypersecretory production of glucose oxidase in Pichia pastoris through combinatorial engineering of protein properties, synthesis, and secretion.

Glucose oxidase (EC 1.1.3.4, GOD) is a widely used industrial enzyme. To construct a GOD-hyperproducing Pichia pastoris strain, combinatorial strategies have been applied to improve GOD activity, synthesis, and secretion. First, wild-type GOD was subjected to saturation mutagenesis to obtain an improved variant, MGOD1 (V20W/T30S), with 1.7-fold higher kcat /KM . Subsequently, efficient signal peptides were screened, and the copy number of MGOD1 was optimized to generate a high-producing strain, 8GM1, containing eight copies of AOX1 promoter-GAS1 signal peptide-MGOD1 expression cassette. Finally, the vesicle trafficking of 8GM1 was engineered to obtain the hyperproducing strain G1EeSe co-expressing the trafficking components EES and SEC. 22, and the EES gene (PAS_chr3_0685) was found to facilitate both protein secretion and production for the first time. Using these strategies, GOD secretion was enhanced 65.2-fold. In the 5-L bioreactor, conventional fed-batch fermentation without any process optimization resulted in up to 7223.0 U/mL extracellular GOD activity (3.3-fold higher than the highest level reported to date), with almost only GOD in the fermentation supernatant at a protein concentration of 30.7 g/L. Therefore, a GOD hyperproducing strain for industrial applications was developed, and this successful case can provide a valuable reference for the construction of high-producing strains for other industrial enzymes.

The small protein MntS evolved from a signal peptide and acquired a novel function regulating manganese homeostasis in Escherichia coli.

Small proteins (<50 amino acids) are emerging as ubiquitous and important regulators in organisms ranging from bacteria to humans, where they commonly bind to and regulate larger proteins during stress responses. However, fundamental aspects of small proteins, such as their molecular mechanism of action, downregulation after they are no longer needed, and their evolutionary provenance, are poorly understood. Here, we show that the MntS small protein involved in manganese (Mn) homeostasis binds and inhibits the MntP Mn transporter. Mn is crucial for bacterial survival in stressful environments but is toxic in excess. Thus, Mn transport is tightly controlled at multiple levels to maintain optimal Mn levels. The small protein MntS adds a new level of regulation for Mn transporters, beyond the known transcriptional and post-transcriptional control. We also found that MntS binds to itself in the presence of Mn, providing a possible mechanism of downregulating MntS activity to terminate its inhibition of MntP Mn export. MntS is homologous to the signal peptide of SitA, the periplasmic metal-binding subunit of a Mn importer. Remarkably, the homologous signal peptide regions can substitute for MntS, demonstrating a functional relationship between MntS and these signal peptides. Conserved gene neighborhoods support that MntS evolved from the signal peptide of an ancestral SitA protein, acquiring a life of its own with a distinct function in Mn homeostasis.

Development of an efficient protein expression system in the thermophilic fungus Myceliophthora thermophila.

BACKGROUND: Thermophilic fungus Myceliophthora thermophila has been widely used in industrial applications due to its ability to produce various enzymes. However, the lack of an efficient protein expression system has limited its biotechnological applications. RESULTS: In this study, using a laccase gene reporting system, we developed an efficient protein expression system in M. thermophila through the selection of strong constitutive promoters, 5'UTRs and signal peptides. The expression of the laccase was confirmed by enzyme activity assays. The results showed that the Mtpdc promoter (Ppdc) was able to drive high-level expression of the target protein in M. thermophila. Manipulation of the 5'UTR also has significant effects on protein expression and secretion. The best 5'UTR (NCA-7d) was identified. The transformant containing the laccase gene under the Mtpdc promoter, NCA-7d 5'UTR and its own signal peptide with the highest laccase activity (1708 U/L) was obtained. In addition, the expression system was stable and could be used for the production of various proteins, including homologous proteins like MtCbh-1, MtGh5-1, MtLPMO9B, and MtEpl1, as well as a glucoamylase from Trichoderma reesei. CONCLUSIONS: An efficient protein expression system was established in M. thermophila for the production of various proteins. This study provides a valuable tool for protein production in M. thermophila and expands its potential for biotechnological applications.