Cross-linking Compounds Research Articles

Next Generation Techniques for Determination of Protein-Protein Interactions: Beyond the Crystal Structure.

We discuss recent advancements in structural biology methods for investigating sites of protein-protein interactions. We will inform readers outside the field of structural biology about techniques beyond crystallography, and how these different technologies can be utilized for drug development. Advancements in cryo-electron microscopy (cryoEM) and micro-electron diffraction (microED) may change how we view atomic resolution structural biology, such that well-ordered macrocrystals of protein complexes are not required for interface identification. However, some drug discovery applications, such as lead peptide compound generation, may not require atomic resolution; mass spectrometry techniques can provide an expedited path to generation of lead compounds. New crosslinking compounds, more user-friendly data analysis, and novel protocols such as protein painting can advance drug discovery programs, even in the absence of atomic resolution structural data. Finally, artificial intelligence and machine learning methods, while never truly replacing experimental methods, may provide rational ways to stratify potential druggable regions identified with mass spectrometry into higher and lower priority candidates. Electron diffraction of nanocrystals combines the benefits of both x-ray diffraction and cryoEM, and may prove to be the next generation of atomic resolution protein-protein interface identification. However, in situations such as peptide drug discovery, mass spectrometry techniques supported by advancements in computational methods will likely prove sufficient to support drug discovery efforts. In addition, these methods can be significantly faster than any crystallographic or cryoEM methods for identification of interacting regions.

Improvement in volume resistivity and morphology of a blend of polyolefin elastomer with linear low-density polyethylene

A silane moisture-cured polyolefin elastomer/linear low-density polyethylene (LLDPE) blend was prepared through a two-step silane-grafting method (Sioplas Process) in an industrial scale twin-screw extruder. The silane-grafted compound was used to make wire and cable coatings. In this work, the effect of some interactive parameters on quality of the products prepared by the above method has been studied, while so far, there have been less experimental investigations. The volume resistivity of cross-linked compound was changed from 2.96 × 1014 to 7.41 × 1014 Ω cm with increasing LLDPE component by maximum 10 wt%. Surface morphology of the product was corrected with reduction in benzoyl peroxide (BPO) concentration from 0.2 wt% to 0.13 wt%. BPO at this level acted as an initiator in grafting reaction of vinyl trimethoxysilane. The curing condition and specimen preparation method by injection molding and/or extrusion were factors which influenced the hot-set test results at 200 °C. The results of tensile and elongation studies showed a maximum value of 9 MPa and 397% for the tests, after 6 h curing. With increases in curing time at a specified temperature, the gel content of the cross-linked compound was increased and reached its maximum value. The maximum gel content values were found to be approximately 60%, 80%, and 82% at temperatures of 25, 60, and 85 °C, respectively. The hardness, density, and tear strength of the samples did not vary significantly with the curing temperature.

Synthesis and functionalization of carbon nanotubes and nanospheres as a support for the immobilization of an enzyme extract from the mushroom Trametes versicolor

In this work, an enzyme extract immobilization over carbon nanomaterials is presented; that is, carbon nanospheres (CNSs), and multi-walled carbon nanotubes (CNTs), using covalent bonds with glutaraldehyde as a crosslinking compound. Carbon nanostructures were obtained by chemical vapor deposition using an iron–cobalt powder alloy prepared by the sol–gel process and using silica powder as support. Acetylene gas was used as carbon carrier to prepare both the CNTs and CNSs. Both nanostructures were oxidized with an acid treatment in order to add functional groups to the surface and subsequently immobilize the enzymes. Carbon nanostructures were characterized using scanning electron microscopy, infrared spectroscopy and Raman spectroscopy to confirm oxidation, immobilization and structural properties. Electrochemical impedance spectroscopy revealed that carbon nanostructures improve the electron transfer of the electrode. Finally, the cyclic voltammetry provided information about the redox process before and after immobilization. Even more, the catalytic activity of the enzymes extract was evaluated showing that CNSs are the best support.

Organization of ATP-gated P2X1 receptor intracellular termini in apo and desensitized states.

The human P2X1 receptor (hP2X1R) is a trimeric ligand-gated ion channel opened by extracellular ATP. The intracellular amino and carboxyl termini play significant roles in determining the time-course and regulation of channel gating-for example, the C terminus regulates recovery from the desensitized state following agonist washout. This suggests that the intracellular regions of the channel have distinct structural features. Studies on the hP2X3R have shown that the intracellular regions associate to form a cytoplasmic cap in the open state of the channel. However, intracellular features could not be resolved in the agonist-free apo and ATP-bound desensitized structures. Here we investigate the organization of the intracellular regions of hP2X1R in the apo and ATP-bound desensitized states following expression in HEK293 cells. We couple cysteine scanning mutagenesis of residues R25-G30 and H355-R360 with the use of bi-functional cysteine reactive cross-linking compounds of different lengths (MTS-2-MTS, BMB, and BM(PEG)2), which we use as molecular calipers. If two cysteine residues come into close proximity, we predict they will be cross-linked and result in ∼66% of the receptor subunits running on a Western blot as dimers. In the control construct (C349A) that removed the free cysteine C349, and some cysteine-containing mutants, cross-linker treatment does not result in dimerization. However, we detect efficient dimerization for R25C, G30C, P358C, K359C, and R360C. This selective pattern indicates that there is structural organization to these regions in the apo and desensitized states in a native membrane environment. The existence of such precap (apo) and postcap (desensitized) organization of the intracellular domains would facilitate efficient gating of the channel.

Spatial functionalization of graphene powder using 1,4-dichlorobutane on ceramic substrate

The production of spatial structures on the ceramic substrates based on graphene powder cross-linked with 1,4-dichlorobutane under different parameters was studied. The influence of the type of environment, the concentration of the crosslinking compound and the time of crosslinking on the functionalization mechanism were determined. It has been shown that there is no significant effect of the solvent polarity on the attained degradation level (a measure of the degree of cross-linking of graphene flakes expressed as ID/IG parameter). Finally, the maximum defect volume was achieved with 120 h of functionalization, with a concentration of 90 mM, regardless of the environment in which the reactions took place. In this case the ID/IG parameter was close to 1.0, which contrasts the value obtained for pure powder where ID/IG was 0.77.

3D freeform printing of silk fibroin

Freeform fabrication has emerged as a key direction in printing biologically-relevant materials and structures. With this emerging technology, complex structures with microscale resolution can be created in arbitrary geometries and without the limitations found in traditional bottom-up or top-down additive manufacturing methods. Recent advances in freeform printing have used the physical properties of microparticle-based granular gels as a medium for the submerged extrusion of bioinks. However, most of these techniques require post-processing or crosslinking for the removal of the printed structures (Miller et al., 2015; Jin et al., 2016) [1,2]. In this communication, we introduce a novel method for the one-step gelation of silk fibroin within a suspension of synthetic nanoclay (Laponite) and polyethylene glycol (PEG). Silk fibroin has been used as a biopolymer for bioprinting in several contexts, but chemical or enzymatic additives or bulking agents are needed to stabilize 3D structures. Our method requires no post-processing of printed structures and allows for in situ physical crosslinking of pure aqueous silk fibroin into arbitrary geometries produced through freeform 3D printing. Statement of Significance3D bioprinting has emerged as a technology that can produce biologically relevant structures in defined geometries with microscale resolution. Techniques for fabrication of free-standing structures by printing into granular gel media has been demonstrated previously, however, these methods require crosslinking agents and post-processing steps on printed structures. Our method utilizes one-step gelation of silk fibroin within a suspension of synthetic nanoclay (Laponite), with no need for additional crosslinking compounds or post processing of the material. This new method allows for in situ physical crosslinking of pure aqueous silk fibroin into defined geometries produced through freeform 3D printing.

The Effect of Tri Poly Phosphate Concentration towards Chitosan-Alginate Micro-particle Characterization and Dissolution Process in Acidic Medium

Chitosan-Alginate micro-particles can serve as coating to the drug through the encapsulation process for controlled release. The addition of Tri Poly Phosphate would change the characterization of Chitosan-Alginate micro-particles because of the process of cross linking. This study aims to determine the characterization of micro-particles of Chitosan-Alginate based on particle size distribution, particle surface morphology and dissolution process in acid medium. The method used to make micro-particles of Chitosan-Alginate is using spray drying, thus the micro-particles can be made by mixing Chitosan-Alginate with a weight ratio of 1: 1, 1: 2, 2: 1 then mixed the crosslinking compound Tri Poly Phosphate (TPP) with a concentration of 0.1%, 0.3%, 0.5%, and added emulsifier Tween 80 3% v / v, stirred at a speed of 20,000 rpm for 10 minutes then inserted into the spray dryer to form micro-particles. The powder of micro-particles then analysed by Scanning Electron Microscopy (SEM) and performed in vitro of stomach acid solution using acidic medium for the test of dissolution. Based on the research that has been done, 0.1% TPP concentration of Chitosan-Alginate ratio of 2: 1 can produce the most uniform size distribution with a ratio of 40.54%. The biggest dissolution of micro-particles obtained from the samples with Chitosan-Alginate ratio of 2: 1 with a concentration of 0.1% TPP was 80.8%.

Interrelation of Hierarchy of Structures at Comparison of Linear and Cross-linked Polymer Compounds

The comparison of behavior of a linear polymer compounds and cross-linked networks are carried out. It is shown, that the correspondence between rheological behavior of a linear polymer compound and macroscopic behavior of its cross-linked analog is determined by topology of structures describing a hydrogel as qualitatively new object as well as by the type of polymer – solvent interactions. In this situation swelling degree is completely described by the Katchalsky – Lifson’s theory. Moreover it is possible to predict microscopic behavior of macromolecular chains on the base of experimental studies of macroscopic specimens of the gel. Such investigations may be carried out in direct way for organogels. When the gel is charged (polyelectrolyte effect takes place) it is necessary to make some correction. The fact is due to so called effect of concentration redistribution, which occurs when surface of the gel acts as a membrane. In such situation the concentration of low-molecular salt inside gel may be quite more low then the concentration of the salt outside specimen. Thus in the solutions of ionogenic salts real behavior of the specimen is determined not average concentration of salt, but the real concentration of salt inside of the gel. Measuring this concentration experimentally it is possible to carried out investigations of charged macromolecular chains on the base of macroscopic specimens too.

Simple Quantification of Pentosidine in Human Urine and Plasma by High-Performance Liquid Chromatography.

Pentosidine is an advanced glycation end-product (AGE) and fluorescent cross-link compound. A simple high-performance liquid chromatographic (HPLC) method was developed for the detection and quantification of pentosidine in human urine and plasma. The mobile phase used a gradient system to improve separation of pentosidine from endogenous peaks, and chromatograms were monitored by fluorescent detector set at excitation and emission wavelengths of 328 and 378 nm, respectively. The retention time for pentosidine was 24.3 min and the lower limits of quantification (LLOQ) in human urine and plasma were 1 nM. The intraday assay precisions (coefficients of variation) were generally low and found to be in the range of 5.19–7.49% and 4.96–8.78% for human urine and plasma, respectively. The corresponding values of the interday assay precisions were 9.45% and 4.27%. Accuracies (relative errors) ranged from 87.9% to 115%. Pentosidine was stable in a range of pH solutions, human urine, and plasma. In summary, this HPLC method can be applied in future preclinical and clinical evaluation of pentosidine in the diabetic patients.

Contribution of crosslinking products in the flavour enhancer processing: the new concept of Maillard peptide in sensory characteristics of Maillard reaction systems.

In this study, the flavour-enhancing properties of the Maillard reaction products (MRPs) for different systems consisted of different peptides (sunflower, SFP; corn, CP and soyabean SP) with, xylose and cysteine were investigated. Maillard systems from peptides of sunflower, corn and soyabean with xylose and cysteine were designated as PXC, MCP and MSP, respectively. The Maillard systems were prepared at pH of 7.4 using temperature of 120C for 2 h. Results showed that all systems were significantly different in all sensory attributes. The highest scores for mouthfulness and continuity were observed for MCP with the lowest peptides distribution between 1000 and 5000Da, known as Maillard peptide. This revealed that the MCP with the lowest Maillard peptide content had the strongest "Kokumi" effect compared to the other MRPsand demonstrated that "kokumi effect" of MRPs was contributed by not only the "Maillard peptide" defined by the molecular weight (1000-5000Da). Results on sensory evaluation after fractionation of PXC followed by enzymatic hydrolysis showed no significant differences between PXC, P-PXC and their hydrolysates. This observation therefore confirmed that the presence of other contributors attributed to the "Kokumi" effect rather than the Maillard peptide. It can be deduced that the unhydrolyzed crosslinking products might have contributed to the "Kokumi" effect of MRPs. The structures of four probable crosslinking compounds were proposed and the findings have provided new insights in the sensory characteristics of xylose, cysteine and sunflower peptide MRPs.

Pembuatan Nanopartikel Kitosan-Fe3O4 secara Kopresipitasi Ex-Situ menggunakan Tripolyphosphate/Sulfat sebagai Crosslinker dan Karakterisasinya Menggunakan XRD

Magnetic nanoparticles of Fe 3 O 4 was synthesized in this research by using ex - situ copresipitation method followed by surface modification with crosslinked chitosan along with trypolyphospate (TPP) and sulfate crosslinking agents. The Fe 3 O 4 materials were formed from FeCl 2 .H 2 O and FeCl 3 .6H 2 O with 1:2 ratio under alkaline condition. Surface modification processes were conducted by mixing chitosan solution with purified Fe 3 O 4 yielding a ferrogel solution as a product. Here, the crosslinker agents, TPP and sulfate, were added to the mixture to make a crosslinking compounds. In these processes, the chitosan-Fe 3 O 4 crystallite size were affected by composition ratio and crosslinking time which will be proven by using x-ray diffraction (XRD) and modified Debye-Scherrer methods. The results showed that, on highest ratios between chitosan and Fe 3 O 4 as well as TPP/sulfate crosslinker, gave the smallest size of crystallite of chitosan-Fe 3 O 4 . Furthermore, the more time of crosslinking process caused crystallite size of chitosan-Fe 3 O 4 became much smaller.

Molecular Interaction Studies of Chitosan Cross-linked Compounds as Drug Delivery Substrate for Anticancer Agents

Chitosan is known for its absorption and adhesion property and it is a non-toxic biodegradable hetero polymer. It has a strong affinity for water and high degree of solubility in acidic medium. In addition, chitosan hydrogels showed low mechanical strength and minimum ability to control the delivery of encapsulated compounds. And hence, in this investigation a set of compounds cross linked with Chitosan was screened for anticancer agent using computational technique (molecular docking) against NOS enzyme (PDB ID: 4NOS). The result was interesting as majority of the compounds screened turned up with favorable molecular interaction and binding affinity as evidenced from the docking score. Furthermore the molecular interaction analysis represents the cross linked compounds possessed heavy molecular interaction at the active site residue of the enzyme. Thus, Chitosan cross linked compounds target the specific active site residue and hence can be used in future for drug delivery.

CRISPR-Cas9-based target validation for p53-reactivating model compounds.

Inactivation of the p53 tumor suppressor by Mdm2 is one of the most frequent events in cancer, so compounds targeting the p53-Mdm2 interaction are promising for cancer therapy. Mechanisms conferring resistance to p53-reactivating compounds are largely unknown. Here we show using CRISPR-Cas9–based target validation in lung and colorectal cancer that the activity of nutlin, which blocks the p53-binding pocket of Mdm2, strictly depends on functional p53. In contrast, sensitivity to the drug RITA, which binds the Mdm2-interacting N terminus of p53, correlates with induction of DNA damage. Cells with primary or acquired RITA resistance display cross-resistance to DNA crosslinking compounds such as cisplatin and show increased DNA cross-link repair. Inhibition of FancD2 by RNA interference or pharmacological mTOR inhibitors restores RITA sensitivity. The therapeutic response to p53-reactivating compounds is therefore limited by compound-specific resistance mechanisms that can be resolved by CRISPR-Cas9-based target validation and should be considered when allocating patients to p53-reactivating treatments.

Fibulin-4 E57K Knock-in Mice Recapitulate Cutaneous, Vascular and Skeletal Defects of Recessive Cutis Laxa 1B with both Elastic Fiber and Collagen Fibril Abnormalities

Fibulin-4 is an extracellular matrix protein essential for elastic fiber formation. Frameshift and missense mutations in the fibulin-4 gene (EFEMP2/FBLN4) cause autosomal recessive cutis laxa (ARCL) 1B, characterized by loose skin, aortic aneurysm, arterial tortuosity, lung emphysema, and skeletal abnormalities. Homozygous missense mutations in FBLN4 are a prevalent cause of ARCL 1B. Here we generated a knock-in mouse strain bearing a recurrent fibulin-4 E57K homozygous missense mutation. The mutant mice survived into adulthood and displayed abnormalities in multiple organ systems, including loose skin, bent forelimb, aortic aneurysm, tortuous artery, and pulmonary emphysema. Biochemical studies of dermal fibroblasts showed that fibulin-4 E57K mutant protein was produced but was prone to dimer formation and inefficiently secreted, thereby triggering an endoplasmic reticulum stress response. Immunohistochemistry detected a low level of fibulin-4 E57K protein in the knock-in skin along with altered expression of selected elastic fiber components. Processing of a precursor to mature lysyl oxidase, an enzyme involved in cross-linking of elastin and collagen, was compromised. The knock-in skin had a reduced level of desmosine, an elastin-specific cross-link compound, and ultrastructurally abnormal elastic fibers. Surprisingly, structurally aberrant collagen fibrils and altered organization into fibers were characteristics of the knock-in dermis and forelimb tendons. Type I collagen extracted from the knock-in skin had decreased amounts of covalent intermolecular cross-links, which could contribute to the collagen fibril abnormalities. Our studies provide the first evidence that fibulin-4 plays a role in regulating collagen fibril assembly and offer a preclinical platform for developing treatments for ARCL 1B.

Synthesis Photosical Properties of Silicon-Containing Cross-Linked Polymer

Organic light-emitting materials in Organic Light-emitting Diodes(OLED) reserch in a very important posotion, the quality of materials directly affect the level of luminous efficiency of the device. We chose benzene 2,6-alkynyl, respectively, and tetrakis (4-bromophenyl) silane, tetrakis (3-bromophenyl) silane synthesis of new cross-linked polymer, the structure was characterized by solid NMR, by fluorescence chromatography UV crosslinking compound characterization of chromatographic performance in photophysical aspects may choose to add a new organic light-emitting material.

ATP-dependent Conformational Changes Trigger Substrate Capture and Release by an ECF-type Biotin Transporter

Energy-coupling factor (ECF) transporters for vitamins and metal ions in prokaryotes consist of two ATP-binding cassette-type ATPases, a substrate-specific transmembrane protein (S component) and a transmembrane protein (T component) that physically interacts with the ATPases and the S component. The mechanism of ECF transporters was analyzed upon reconstitution of a bacterial biotin transporter into phospholipid bilayer nanodiscs. ATPase activity was not stimulated by biotin and was only moderately reduced by vanadate. A non-hydrolyzable ATP analog was a competitive inhibitor. As evidenced by cross-linking of monocysteine variants and by site-specific spin labeling of the Q-helix followed by EPR-based interspin distance analyses, closure and reopening of the ATPase dimer (BioM2) was a consequence of ATP binding and hydrolysis, respectively. A previously suggested role of a stretch of small hydrophobic amino acid residues within the first transmembrane segment of the S units for S unit/T unit interactions was structurally and functionally confirmed for the biotin transporter. Cross-linking of this segment in BioY (S) using homobifunctional thiol-reactive reagents to a coupling helix of BioN (T) indicated a reorientation rather than a disruption of the BioY/BioN interface during catalysis. Fluorescence emission of BioY labeled with an environmentally sensitive fluorophore was compatible with an ATP-induced reorientation and consistent with a hypothesized toppling mechanism. As demonstrated by [(3)H]biotin capture assays, ATP binding stimulated substrate capture by the transporter, and subsequent ATP hydrolysis led to substrate release. Our study represents the first experimental insight into the individual steps during the catalytic cycle of an ECF transporter in a lipid environment.

Effect of thermal treatment on the flavor generation from Maillard reaction of xylose and chicken peptide

In this research, effect of the thermal treatment on the flavor generation from Maillard reaction of xylose and chicken peptide was studied. In summary, high temperature (>100 °C) could remarkably increase the formation of meaty aroma generated by xylose and chicken peptide through a Maillard reaction system, while lower temperature and longer heating tended to generate a broth-like taste (i.e., umami and kokumi). Pyrazines were the major contributors to the nutty/roast and basic meaty aroma in the Maillard reaction products (MRP), while the Glu released under the low temperature heating was considered an important contributor to the umami taste of MRPs. The low molecular weight peptide (<500 Da) was considered as the main contributor of the generation of pyrazines and 2-furfurylpyrrole, which could be due to the high reaction activity of the amidogen in these compounds. In addition, cross-linking compounds with molecular weight >1000 Da formed from peptides <500 Da in the low temperature heating might be involved in formation of important kokumi-active compounds, while the high molecular compounds MRPs >3000 Da were very likely responsible for the bitter taste.

Guided ion beam and computational studies of the decomposition of a model thiourea protein cross-linker.

The dissociation of protonated methyl-d3 thiourea-4-butyric acid methyl amide (1), a model of thiourea-based protein cross-linking compounds, is examined both experimentally and computationally. Using a guided ion beam tandem mass spectrometer (GIBMS), the threshold collision-induced dissociation (TCID) of [1 + H](+) with Xe is examined as a function of collision energy. Analysis of the kinetic energy-dependent CID cross sections provides the 0 K barriers for four primary and four secondary dissociation pathways, after accounting for competition between channels, sequential dissociations, unimolecular decay rates, internal energy of reactant ions, and multiple ion-neutral collisions. Computations are used to explore the pathways for the various processes and elucidation of their rate-limiting transition states. These results indicate that dissociation is initiated by migration of the excess proton from sulfur to one of three nitrogen atoms in 1, similar to the "mobile proton" model of peptide fragmentation. The computational energies for the rate-limiting transition states are generally in good agreement with the experimentally derived threshold energies, with MP2(full)/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) results being particularly favorable. This good comparison validates the mechanisms explored theoretically and allows identification of the structures of the various product ions and neutrals.

Stabilization of collagen through bioconversion: An insight in protein–protein interaction

Collagen is a natural protein, which is used as a vital biomaterial in tissue engineering. The major concern about native collagen is lack of its thermal stability and weak resistance to proteolytic degradation. In this scenario, the crosslinking compounds used for stabilization of collagen are mostly of chemical nature and exhibit toxicity. The enzyme mediated crosslinking of collagen provides a novel alternative, nontoxic method for stabilization. In this study, aldehyde forming enzyme (AFE) is used in the bioconversion of hydroxylmethyl groups of collagen to formyl groups that results in the formation of peptidyl aldehyde. The resulted peptidyl aldehyde interacts with bipolar ions of basic amino acid residues of collagen. Further interaction leads to the formation of conjugated double bonds (aldol condensation involving the aldehyde group of peptidyl aldehyde) within the collagen. The enzyme modified collagen matrices have shown an increase in the denaturation temperature, when compared with native collagen. Enzyme modified collagen membranes exhibit resistance toward collagenolytic activity. Moreover, they exhibited a nontoxic nature. The catalytic activity of AFE on collagen as a substrate establishes an efficient modification, which enhances the structural stability of collagen. This finds new avenues in the context of protein-protein stabilization and discovers paramount application in tissue engineering.

On the mechanism of deposit formation during thermal oxidation of mineral diesel and diesel/biodiesel blends under accelerated conditions

The influence of temperature and time on the formation process of solid deposits after accelerated degradation of mineral diesel (B0) and a biodiesel blend (diesel/rapeseed methyl ester blend (B20)) is studied in model experiments. We show that the formation of deposits strongly depends on the temperature to which the fuel is exposed. Heat-treatment of the fuel samples at above 140°C for longer than 6h under constant air flow leads to the formation of colored, homogeneous solutions containing polar, high molecular weight oxygenated fuel products (mOFP). The formed mOFP are precipitated in n-heptane and subsequently analyzed. Correlations between temperature and duration of the heat-treatment and the amount of precipitated mOFP as well as the acidity of the fuels and spectroscopic data of the fuels are elucidated. Upon heating of isolated mOFP precursors at temperatures above 150°C solid fuel deposits are formed. Analysis by size exclusion chromatography shows that, depending on temperature and reaction time, the molecular weight increases, the molecule become branched and ultimately cross-linked compounds are formed. At temperatures of up to 200°C the B20 deposits remain completely soluble in a common solvent with an intermediate polarity such as tetrahydrofuran (THF), independent of reaction time. With increasing temperature and duration of the heat exposure the solubility becomes more and more reduced until eventually complete insolubility is reached. A comparison between heat treated model compounds and deposits isolated after completion of engine testing experiments is made. The implications of the improved understanding of the deposit formation process onto new design rules for possible next-generation injector designs are discussed.