Substrate Movement Research Articles

Rock and roll: experiments on substrate movement and coral settlement

Rubble is ubiquitous on coral reefs and can aggregate into fields, forming a significant component of the reef substrate. Rubble fields often remain unconsolidated, with the component rubble pieces subject to movement that is dependent on hydrodynamic forcing, rubble size, shape, and other factors. Settlement of corals to rubble fields has long been assumed, but the dynamic movement of rubble pieces has been presumed to deter settlement and is thought to contribute to high post-settlement mortality. Rubble often forms on coral reefs following severe disturbances, and is predicted to increase under climate change, with the potential to impact settlement and recruitment-dependent recovery processes. Through a series of laboratory and field experiments, we demonstrate that corals from broadcast spawning species on the Great Barrier Reef will settle on unstable substrates, even those in constant motion. We also observed more coral spat on settlement tiles suspended in the water column than those fixed to the reef using a common approach to censusing settlement. Sampling of natural rubble on the reef 50 days after a mass-spawning event confirmed the presence of similar numbers of coral settlers on rubble and on tiles fixed to the reef. These results suggest that rubble fields are places of significant settlement for broadcast spawning corals. Suspended tiles were also surprisingly effective in collecting coral settlers, demonstrating that a change in sampling protocol can produce significant variation in settlement data and strengthening the argument for standardisation of settlement-monitoring protocols, particularly at a time of growing need for reliable metrics. These results also suggest that movement of rubble is not precluding settlement outright, but rather post-settlement processes (i.e. competition, predation, shading or burial by shifting rubble) are limiting recruitment to rubble patches. Consequently, rubble stabilisation may increase the survival of spat that have settled in these environments.

Additive manufacturing of quartz glass using coaxial wire feeding technology

This study explores the process of coaxial wire feeding and melting for quartz additive manufacturing using Direct Energy Deposition (DED). Quartz glass, valued for its excellent mechanical, optical, and thermal properties, is widely used in semiconductor, aerospace, and optical communication industries. Additive manufacturing offers advantages such as cost-effective materials, rapid formation, and customization of structures. However, current methods for additive manufacturing of quartz glass often result in low quality or require complex post-processing, particularly for optical components. In optics, there is a demand for high-quality, straightforward processes, and custom-shaped quartz glass. This article presents a coaxial wire feeding mechanism wherein quartz wire is fed vertically downward along the optical axis into the melting zone created by a CO2 laser, facilitating smooth material deposition onto the molten zone’s surface. Laser power, wire feeding speed, substrate movement speed, and defocusing distance are key factors influencing the quality of single-layer quartz glass formation. Positive defocusing was found to have a beneficial impact on single-layer quartz glass additive manufacturing. Subsequently, optimized process parameters enabled the production of uniformly cross-sectional single-layer quartz glass. Microscopic morphology analysis and temperature field simulation were conducted on the experimental results before and after optimization. Finally, complex quartz glass structures were additively manufactured using the optimized experimental parameters. Results demonstrate that coaxial wire feeding technology has significant potential for achieving uniform dimensions and complex structures in quartz glass additive manufacturing.

A Review of P-Glycoprotein Function and Regulation in Fish

The teleost ATP Binding Cassette (ABC) transporter P-glycoprotein (P-gp) is an active transmembrane transporter that plays a pivotal role in facilitating the movement of both endogenous and xenobiotic substrates (moderately hydrophobic and amphipathic compounds) across cell membranes. P-gp exhibits substrate specificity often shared with other ABC transporters and solute carrier proteins, thereby ensuring the maintenance of chemical homeostasis within cells. These transporters are integral to chemical defense systems in fish, as they actively expel a wide range of substrates, primarily unmodified compounds, from cells. This transport process assists in preventing chemical absorption (e.g., intestine), safeguarding sensitive tissues (e.g., brain and gonads), and effectively excreting substances (e.g., liver and kidney). Upregulated P-gp export activity in aquatic animals results in the multi-xenobiotic resistance (MXR) phenotype that plays an essential protective role in survival in contaminated environments. Pollutants inhibiting P-gp are termed chemosensitizers and heighten fish sensitivity to toxic P-gp substrates. While the known intrinsic functions of P-gp in fish encompass steroid hormone and bile acid processing, relatively little attention has been given to endogenous substrates and inhibitors. Fish P-glycoprotein regulation is orchestrated by pivotal nuclear transcription factors, including pregnane X receptor (PXR) and nuclear factor erythroid 2-related factor 2 (Nrf2). This comprehensive review provides profound insights into P-gp’s significance across diverse fish species, contributing to an enhanced understanding of fish physiology, evolution, and toxicology, and provides information with potential applications, such as environmental monitoring.

Bioprinting with 3-µm laser pulses

Laser-Induced Forward Transfer (LIFT) technology has now found its wide application in the field of bioprinting. Laser bioprinting allows the spatial transfer of living micro-objects (cells, microorganisms) in a gel medium using the energy of a laser pulse. Typically, to effectively absorb laser pulse energy, a thin metal layer is deposited on the donor substrate. The disadvantages of this approach are the appearance of cytotoxic metal nanoparticles during laser bioprinting and the need for continuous movement of the donor substrate. The article proposes a method that allows moving to absorption film-free bioprinting using Q-switched Er:YAG nanosecond laser operating at a wavelength of 2.94 µm. This wavelength is effectively absorbed in a top ten-micron hydrogel layer, allowing for a cleaner (without nanoparticles from absorbing layer) and simpler (without the need to constantly move the donor substrate from pulse to pulse) laser bioprinting process. Using strain Escherichia coli as an example, the suitability of the proposed method for successful laser bioprinting is demonstrated. A model describing in detail the processes occurring in the hydrogel layer is presented.

Classification of SLC family-related genes involved in ferroptosis predicts lung cancer prognosis and immunotherapy response

Lung adenocarcinoma, the most frequent type of lung cancer, is the leading cause of cancer-related deaths worldwide. Ferroptosis, controlled cell death that involves a high degree of iron-dependent lipid peroxidation, has been linked to tumor therapy sensitivity, patient prognosis, and cancer development. The solute carrier superfamily has over 400 members and comprises the largest class of transporters in the human genome. Solute carrier proteins can facilitate the movement of different substrates across biological membranes, which is crucial for physiological activities, including ferroptosis. Here, we developed a new model to further explore the role of the solute carrier family in ferroptosis in the lung adenocarcinoma immunological milieu. We used consensus clustering to classify patients with lung cancer into two subgroups (cluster1 and cluster2). Patients in the cluster1 subtype had a better prognosis and higher immune cell infiltration ratios than those in the cluster2 subtype. Furthermore, to evaluate the prognosis, the immune cell infiltration ratio, and the medication sensitivity of patients with lung adenocarcinoma, we developed gene scores related to the solute carrier family. In conclusion, we successfully developed a model incorporating the solute carrier family and ferroptosis to predict survival and the impact of immunotherapy on patients with lung cancer.

Structure of the Blood Brain Barrier and its Role in the Transporters for the Movement of Substrates across the Barriers.

For the brain to operate normally and to develop with structural integrity in addition to neuronal function, blood-brain barrier present in brain capillaries serves as a vital barrier mechanism. In addition to the transport barrier created by membranes, transporters, and vesicular processes, the structure and function of the BBB are summarised. The physical barrier is created by endothelial tight junctions. The permeability and transport of molecules between extracellular fluid and plasma are constrained by the presence of tight junctions between neighbouring endothelial cells. Each solute must pass through both membranes in the luminal and abluminal divisions. The functions of the neurovascular unit are described, with special emphasis on the pericytes, microglia, and astrocyte endfeet. The luminal membrane contains five separate facilitative transport mechanisms, each of which is exclusive to a few substrates. Nevertheless, the import of big-branched and aromatic neutral amino acids is facilitated by two key carriers (System L and y+) in the plasma membrane. It is asymmetrically present in both membranes. The sodium pump Na+/K+-ATPase is highly expressed in the abluminal membrane, where many Na+ dependent transport mechanisms move amino acids against its concentration gradient. The trojan horse strategy, which uses molecular tools to bind the medication and its formulations, is also preferred in drug delivery. The BBB's cellular structure, the transport systems unique to each substrate, and the necessity to identify transporters with changes that assist the transfer of various medications have all been changed in the current work. Nevertheless, to rule out the BBB passage for the new class of neuroactive medications, the mixing of traditional pharmacology and nanotechnology needs to be focused on outcomes that show promise.

Impact of Some Toxic Metals on Important ABC Transporters in Soybean (Glycine max L.).

In plants, ATP-binding cassette (ABC) transporters facilitate the movement of substrates across membranes using ATP for growth, development, and defense. Soils contaminated with toxic metals such as cadmium (Cd) and mercury (Hg) might adversely affect the metabolism of plants and humans. In this study, a phylogenetic relationship among soybeans' (Glycine max) ATP binding cassette (GmABCs) and other plant ABCs was analyzed using sequence information, gene structure, chromosomal distribution, and conserved motif-domain. The ontology of GmABCs indicated their active involvement in trans-membrane transport and ATPase activity. Thirty-day-old soybean plants were exposed to 100 μM CdCl2 and 100 μM HgCl2 for 10 days. Physiological and biochemical traits were altered under stress conditions. Compared to Control, GmABC transporter genes were differentially expressed in response to Cd and Hg. The qRT-PCR data showed upregulation of seven ABC transporter genes in response to Cd stress and three were downregulated. On the other hand, Hg stress upregulated four GmABC genes and downregulated six. It could be concluded that most of the ABCB and ABCG subfamily members were actively involved in heavy metal responses. Real-time expression studies suggest the function of specific ABC transporters in Cd and Hg stress response and are helpful in future research to develop stress-tolerant varieties of soybean.

Interaction of systemic drugs causing ocular toxicity with organic cation transporter: an artificial intelligence prediction

Chronic disease patients (cancer, arthritis, cardiovascular diseases) undergo long-term systemic drug treatment. Membrane transporters in ocular barriers could falsely recognize these drugs and allow their trafficking into the eye from systemic circulation. Hence, despite their pharmacological activity, these drugs accumulate and cause toxicity at the non-target site, such as the eye. Since around 40% of clinically used drugs are organic cation in nature, it is essential to understand the role of organic cation transporter (OCT1) in ocular barriers to facilitate the entry of systemic drugs into the eye. We applied machine learning techniques and computer simulation models (molecular dynamics and metadynamics) in the current study to predict the potential OCT1 substrates. Artificial intelligence models were developed using a training dataset of a known substrates and non-substrates of OCT1 and predicted the potential OCT1 substrates from various systemic drugs causing ocular toxicity. Computer simulation studies was performed by developing the OCT1 homology model. Molecular dynamic simulations equilibrated the docked protein-ligand complex. And metadynamics revealed the movement of substrates across the transporter with minimum free energy near the binding pocket. The machine learning model showed an accuracy of about 80% and predicted the potential substrates for OCT1 among systemic drugs causing ocular toxicity – not known earlier, such as cyclophosphamide, bupivacaine, bortezomib, sulphanilamide, tosufloxacin, topiramate, and many more. However, further invitro and invivo studies are required to confirm these predictions. Communicated by Ramaswamy H. Sarma

The chemical neighborhood of cells in a diffusion-limited system.

Microorganisms follow us everywhere, and they will be essential to sustaining long-term human space exploration through applications such as vitamin synthesis, biomining, and more. Establishing a sustainable presence in space therefore requires that we better understand how stress due to the altered physical conditions of spaceflight affects our companion organisms. In microgravity environments such as orbital space stations, microorganisms likely experience the change in gravity primarily through changes in fluid mixing processes. Without sedimentation and density-driven convection, diffusion becomes the primary process governing the movement of growth substrates and wastes for microbial cells in suspension culture. Non-motile cells might therefore develop a substrate-deficient "zone of depletion" and experience stress due to starvation and/or waste build-up. This would in turn impact the concentration-dependent uptake rate of growth substrates and could be the cause of the altered growth rates previously observed in microorganisms in spaceflight and in ground-simulated microgravity. To better understand the extent of these concentration differences and their potential influence on substrate uptake rates, we used both an analytical solution and finite difference method to visualize concentration fields around individual cells. We modeled diffusion, using Fick's Second Law, and nutrient uptake, using Michaelis-Menten kinetics, and assessed how that distribution varies in systems with multiple cells and varied geometries. We determined the radius of the zone of depletion, within which cells had reduced the substrate concentration by 10%, to be 5.04 mm for an individual Escherichia coli cell in the conditions we simulated. However, we saw a synergistic effect with multiple cells near each other: multiple cells in close proximity decreased the surrounding concentration by almost 95% from the initial substrate concentration. Our calculations provide researchers an inside look at suspension culture behavior in the diffusion-limited environment of microgravity at the scale of individual cells.

Scaling maximum spreading of droplet impacting on flexible substrates

We numerically study the impact of a droplet on superhydrophobic flexible plates, aiming to understand how the flexible substrate influences the maximum spreading of the droplet. Compared with the rigid case, the vertical movement of the flexible substrate due to droplet impact reduces the maximum spreading. Besides, the average acceleration $a$ during droplet spreading changes significantly. Arising from energy conservation, we rescale the acceleration $a$ for cases with different bending stiffness $K_B$ and mass ratio $M_r$ . Moreover, through theoretical analysis, we propose a scaling for the droplet's maximum spreading diameter ratio $\beta _{max}$ . In the scaling, based on the derived $a$ , an effective Weber number $We_m$ is well defined, which accounts for the substrate properties without any adjustable parameters. In the ( $\beta _{max}, We_m$ ) plane, the two-dimensional numerical results of different $K_B$ , $M_r$ and rigid cases all collapse into a single curve, as do the experimental and three-dimensional (3-D) results. In particular, the collapsed 3-D data can be well represented by the universal rescaling of $\beta _{max}$ proposed by Lee et al. (J. Fluid Mech., vol. 786, 2016, R4). Furthermore, an a posteriori energy analysis confirms the validation of our a priori scaling law.

Elucidating bacterial lipid transport mechanisms by the envelope-spanning LetAB complex.

Gram-negative bacteria are surrounded by an outer membrane (OM) that protects them against environmental stressors and toxic compounds, and therefore contributes to antibiotic resistance. To generate this barrier, membrane lipids, which are poorly soluble in water, must be transported across the hydrophilic periplasmic space between the inner membrane (IM) and OM. Bacteria have evolved various mechanisms to achieve active transport across the cell envelope. The Let (Lipophilic Envelope-spanning Tunnel) system, comprising LetA and LetB, is one system thought to be involved in maintaining OM integrity. LetB forms a hydrophobic tunnel long enough to span the periplasm, potentially facilitating the sheltered movement of substrates directly between the two membranes. However, it is unclear how lipids are extracted from the IM and transported into LetB, or vice versa. The only known binding partner of LetB is the IM protein, LetA, which belongs to an unstudied family of proteins. We have obtained a ∼3.5 Å cryo-EM structure of the LetAB complex, which reveals that LetA is a pseudodimeric integral inner membrane protein with two cytoplasmic zinc finger domains that interact with each other to stabilize the protein. LetA binds at one end of LetB, where it interacts with the central tunnel of LetB to form a substrate translocation pathway between the two subunits. Our structural and biochemical data reveal a possible substrate-binding site in LetA. LetA also contains a putative proton/ion permeation pathway, lined with ionizable residues that are essential for function, which we hypothesize may play a role in energizing transport. We will discuss how the LetA and LetB subunits come together to facilitate lipid transport, and provide a model for how proteins in the LetA protein family may function.

Mechanism of multimodal substrate translocation in P-glycoprotein.

P-glycoprotein (Pgp) is a highly dynamic ATP-binding cassette transporter that confers cancer multidrug resistance and mediates the bioavailability and pharmacokinetics of many drugs. Structural and biochemical data have provided insights into the binding of diverse compounds, but how they are translocated through the membrane has remained elusive. The biggest hurdle to address this fundamental question has been isolating and characterizing the short-lived transition states during transport. Here, we covalently attached a cyclopeptide substrate to discrete sites of Pgp and performed cryo-EM analysis. A series of substrate-bound Pgp structures in inward- and outward-facing conformations enabled tracing of the substrate passage and revealed how a cascade of conformational changes in transmembrane helix 1 drives substrate movement across the membrane bilayer upon ATP binding. Observation of a double substrate-bound outward-facing conformation suggests that Pgp can co-transport two compounds simultaneously.

Molecular Insights into Substrate Binding of the Outer Membrane Enzyme OmpT

The enzyme OmpT of the outer membrane of Escherichia coli shows proteolytic activity and cleaves peptides and proteins. Using molecular dynamics simulations in a fully hydrated lipid bilayer on a time scale of hundreds of nanoseconds, we draw a detailed atomic picture of substrate recognition in the OmpT-holo enzyme complex. Hydrogen bonds and salt bridges are essential for maintaining the integrity of the active site and play a central role for OmpT in recognizing its substrate. Electrostatic interactions are critical at all stages from approaching the substrate to docking at the active site. Computational alanine scanning based on the Molecular Mechanics Generalized Born Surface Area (MM-GBSA) approach confirms the importance of multiple residues in the active site that form salt bridges. The substrate fluctuates along the axis of the β-barrel, which is associated with oscillations of the binding cleft formed by the residue pairs D210-H212 and D83-D85. Principal component analysis suggests that substrate and protein movements are correlated. We observe the transient presence of putative catalytic water molecules near the active site, which may be involved in the nucleophilic attack on the cleavable peptide bond of the substrate.

Structural basis of SecA-mediated protein translocation

Secretory proteins are cotranslationally or posttranslationally translocated across lipid membranes via a protein-conducting channel named SecY in prokaryotes and Sec61 in eukaryotes. The vast majority of secretory proteins in bacteria are driven through the channel posttranslationally by SecA, a highly conserved ATPase. How a polypeptide chain is moved by SecA through the SecY channel is poorly understood. Here, we report electron cryomicroscopy structures of the active SecA-SecY translocon with a polypeptide substrate. The substrate is captured in different translocation states when clamped by SecA with different nucleotides. Upon binding of an ATP analog, SecA undergoes global conformational changes to push the polypeptide substrate toward the channel in a way similar to how the RecA-like helicases translocate their nucleic acid substrates. The movements of the polypeptide substrates in the SecA-SecY translocon share a similar structural basis to those in the ribosome-SecY complex during cotranslational translocation.

Scalable Electrospinning Methods to Produce High Basis Weight and Uniform Drug Eluting Fibrous Biomaterials

Electrospinning is a process for fabricating nonwoven fibrous materials of versatile composition and form that has shown enormous promise as medical wound dressings, tissue engineered scaffolds, and for pharmaceutical delivery. However, pharmaceutical application and clinical translation of electrospun fibers requires a scalable process to control mass deposition and uniformity in the finished materials. Here, we show that free-surface electrospinning using a stationary wire electrode can generate fiber materials with high productivity and controllable deposition to achieve uniform area density (basis weight) that is relevant for scalable pharmaceutical dosage form production. Using a production-scale instrument, we performed statistically designed optimization experiments to identify a combination of parameters that improved productivity up to 13 g/h. By combining this optimization with process controls for dynamic movement of the electrospinning substrate, we also demonstrate the production of uniform and high area density materials of 50–120 G per square meter. We verified our process by fabricating a triple drug solid dosage form at a high area target density (100 g/m2) that largely showed less than a 10% coefficient of variation in mass or drug content. The process developed here provides a general approach for optimizing different material compositions for high productivity and uniformity, and advances the use of free-surface electrospinning for manufacturing fiber-based biomedical materials.

An internal docking site stabilizes substrate binding to γ-secretase: Analysis by molecular dynamics simulations

Amyloid precursor protein (APP) is cleaved and processed sequentially by γ-secretase yielding amyloid β (Aβ) peptides of different lengths. Longer Aβ peptides are associated with the formation of neurotoxic plaques related to Alzheimer’s disease. Based on the APP substrate-bound structure of γ-secretase, we investigated the enzyme-substrate interaction using molecular dynamics simulations and generated model structures that represent the sequentially cleaved intermediates during the processing reaction. The simulations indicated an internal docking site providing strong enzyme-substrate packing interaction. In the enzyme-substrate complex, it is located close to the region where the helical conformation of the substrate is interrupted and continues toward the active site in an extended conformation. The internal docking site consists of two non-polar pockets that are preferentially filled by large hydrophobic or aromatic substrate side chains to stabilize binding. Placement of smaller residues such as glycine can trigger a shift in the cleavage pattern during the simulations or results in destabilization of substrate binding. The reduced packing by smaller residues also influences the hydration of the active site and the formation of a catalytically active state. The simulations on processed substrate intermediates and a substrate G33I mutation offer an explanation of the experimentally observed relative increase of short Aβ fragment production for this mutation. In addition, studies on a substrate K28A mutation indicate that the internal docking site opposes the tendency of substrate dissociation due to a hydrophobic mismatch at the membrane boundary caused by K28 during processing and substrate movement toward the enzyme active site. The proposed internal docking site could also be useful for the specific design of new γ-secretase modulators.

Predicting the composition of W-B-C coatings sputtered from industrial cylindrical segmented target

Computational methods have been gaining more and more attention as they provide a powerful tool for increasing efficiency in the industrial use of magnetron sputtering and provide results from which experimental work benefits. This work presents an approach for the prediction of the composition and the deposition rate of the coatings. The combination of SDTrimSP and SiMTra provides a framework for the simulation of the deposition process. The chemical composition and the deposition rate of industrially sputtered W-B-C coatings were investigated. An industrial batch coater with a complex geometry was modelled. A cylindrical rotating sputter source was mounted with a segmented target composed of W, B4C, and C segments. The simulated chemical composition and the relative deposition rate were verified and validated by experiments. A correlation between the chemical composition and target segment placement is presented, and the influence of substrate movement on the chemical composition is discussed. A single-axis rotation of the substrate placed on an industrial carousel causes a lowering of the content of tungsten in the coatings in comparison to statically placed substrates in front of the sputter source. The simulations uncover the distinct trajectories of the heavy and light particles. The different transport of particles from target to substrate results in the observed shift of chemical composition and also explains the inherent nanolayering of the non-reactively deposited coatings in the industrial batch coater.

The success of seagrass restoration using Enhalus acoroides seeds is correlated with substrate and hydrodynamic conditions

The extent of seagrass areas and their associated ecosystem functions and services have been declining due to many factors. Seagrass restoration is important to mitigate such declines. Seagrass restoration using seeds can be a viable method due to the high seed availability of some seagrass species and could enhance seagrass resilience to climate change stress. However, this method sometimes has low success rates due to high seed predation and seeds being washed away by wave action or substrate movement. The research was conducted to compare the settlement of Enhalus acoroides seeds and the establishment of seedlings on different sediment types (fine sand, coarse sand, and hard substrate with rubble) combined with different wave exposure levels (high and moderate). This is the first study to observe seed survival and seedling establishment of the tropical seagrass E. acoroides in the wild. On average, 64% of seeds dispersed on fine sand substrate at a moderate exposure site survived and developed into established seedlings by the end of the 40 days observation period, but the survival of seeds dispersed on coarse sand at high exposure and hard substrate at moderate exposure only remained above 50% for up to 3 days, and had declined to 2% and 1.4%, respectively, by day 40. Six years later, surviving E. acoroides sample from the coarse sand and hard substrate both had well-developed rhizomes but fewer roots than the plant from the fine sand site, these rhizome and roots characteristics were likely adaptations to increase anchoring capacity in the specific site. The results indicate that Enhalus seed settlement and seedling establishment can readily occur at sites with fine sand substrate and lower wave exposure; however, additional measures may be required at more exposed sites with mobile or hard substrates until seedlings become established.

Structures of multidrug resistance protein MRP4 reveal basis of substrate specificity

ATP-binding cassette (ABC) transporters are a ubiquitous family of transmembrane proteins which hydrolyze ATP to facilitate the movement of substrates across cell membranes. Despite common architectural features, individual eukaryotic ABC transporters have evolved to transport a broad spectrum of substrates. The multidrug resistance protein MRP4, a member of the MRP sub-family of transporters, has been linked to the efflux of diverse endogenous and exogenous substrates, including prostaglandins, cyclic nucleotides, conjugated steroids, and other chemical scaffolds.

Morphological characteristics of the drop on the travelling substrate with a sharp stepped configuration

The shape of a drop on a partially wetting substrate strongly depends on not only its wetting velocity but also the surface characteristics of the substrate. The unexpected deformation or even breakup of the drop on the heterogeneous travelling substrate is a common phenomenon and needs to be carefully controlled in industrial processes. In this work, we designed an experiment to investigate the morphological characteristics of the drop on the substrate with a sharp stepped configuration. We experimentally studied the entire process of the drop motion on the substrate with a vertical-edged stepped structure and subdivided it into three stages: the collision stage, the inserting stage, and the stretching stage. For the collision stage, substrate velocity was determined as an important factor influencing the tail of the drop before and after the collision. For the inserting stage, it was found that the liquid is transferred to the pre-inserting zone with the reduced gap height due to the squeezing, resulting in an increase in the tail length; it was verified that the tail lengths vary with the position of the stepped frame by assuming that the drop volume is constant and the tail is simplified as a cone. For the stretching stage, three types of drop tails were observed by balancing the motion of the constraint tail in two directions: the one along the substrate movement and the one for anti-constraint of the stepped configuration; we experimentally analyzed the three types of drop tails and found that they are related to the substrate velocity, the gap height, and the height of stepped configuration.