The most recent and advanced class of crystalline and permeable compounds are covalent organic frameworks (COFs). Due to their exceptional qualities, such as their porous structure, high surface area, strong chemical and thermal stabilities, low density, good water stability, luminescent nature, and so on, COFs have seen remarkable growth over the past ten years. COFs have been successfully researched for a number of applications based on these characteristics. The current state of COFs has been reported in this study, with particular attention paid to their design, topology, synthesis, and a variety of biological applications, including drug delivery systems, photodynamic and photothermal therapy, biosensing, bioimaging, etc. Moreover, several miscellaneous applications, such as catalysis, gas storage and separation, photocatalysis, sensors, solar cells, supercapacitors, and 3D printers, have also been explored. It is significant that we have examined current research on COFs with a focus on the biological applications, which are infrequently covered in the literature. Descriptions of the difficulties and prospective outcomes have also been given. The term “covalent organic frameworks” (COFs) refers to a newly discovered category of materials that have a chemically permeable organic structure. In principle, COFs are crystalline materials made of a variety of organic ligands that are covalently connected to create long-range extended structures. The production of crystals in COFs has been accomplished through various approaches that establish the optimal balance between the thermal reversibility of the linking processes and the dynamics of the corresponding crystals . The mai3 silane or a carbon atom. Low densities, a variety of open sites, and high specific surface areas are all visible in the 3D COFs. These excellent properties encourage studies of 3D COFs for drug delivery applications [COFs can be classified either as two-dimensional (2D) or three-dimensional 3D) COFs, depending on the dimensions of the construction blocks. In 2D COFs, the framework of covalent bonds is restricted to 2D sheets. These sheets are then stacked on top of one another to generate a layered structure with overlapping layers that presents periodic columns. This columnar pileup structure offers a novel approach to the construction of an ordered solid network, which is traditionally difficult to achieve through the use of conventional covalent and noncovalent approaches. Since the ordered columns in 2D COFs are able to enhance charge carrier transport in the stacking direction, this indicates that 2D COFs have the potential to be used in various applications. In contrast, the framework of a 2D COF can be extended into the third dimension by using a building block that contains a spD COFs, dications .In addition to the dimension of COFs, they exhibit important features such as structural versatility, easy surface functionalization, pre- and post-synthetic modification, excellent chemical and thermal stabilities, a high surface area, permanent porosity, and low density . The COFWhen compared to other nanosystems, COFs offer several benefits that make them preferable for use in nanomedical applications. These benefits are based on their excellent properties, such as structural periodicity, internal channels, high porosity, and pores . Furthermore, the diversity of COFs can be easily incorporated by carefully choosing and functionalizing organic building blocks. Because of their versatility, COFs can carry and release medications or theranostic agents under precise control, thereby highlighting their special function as diagnostic and therapeutic nanoparticles in synergistic anticancer therapy. Additionally, COFs exhibit exceptional chemical stability and biocompatibility due to their strong covalent bond coupling and nonmetallic characteristics. Recent research has shown that COFs can be used as drug carriers, due to their excellent characteristics, such as high loading efficiency, exceptional release properties, and minimal systemic toxicity. Therefore, using COFs for tumor-targeted anticancer drug delivery in vivo will be quite appealing. Because of their high sensitivity, non-intrusiveness, quick response time, and real-time performance testing, they have also been used in fluorescence imaging to study the roles of biological molecules in a natural microenvironment. Utilizing COFs’ distinctive properties, including the long-range crystal state and eclipsed stacking assembly, they are applied as potential fluorescence imaging agents. Hence, in this part, we have emphasized the uses of COFs for fluorescence imaging ,5,6,7,8.Despite the significant amount of research that has been conducted over the last few years, the application of COFs in cancer treatment is still mostly unknown. Due to the inadequate development of COFs from the perspective of cancer therapies, research into the potential use of COFs in cancer therapy is lacking so far. Therefore, the utilization of COFs in nanomedicine requires additional research. To fully make use of the promising potential of COF-based nanosystems in cancer treatment, one needs to have a sufficient understanding of these systems ,9. As of1O2 generation. As reported, it exhibits a 55.9% photothermal conversion ability [To explore the biomedical applications of COF, covalently grafted porphyrinic PS (POR), VONc@COF-Por, upon visible (red LED) and NIR (808 nm laser) irradiation displayed high  ability . Additio ability . In rece ability   in comparison to simple quercetin molecules. Furthermore, the increased rate of drug loading and prolonged release has also been found due to host–guest interaction and strong hydrogen bonding .1O2 detector, 1,3-diphenylisobenzofuran (DPBF) was used to assess the COF’s photodynamic activity. The absorbance of DPBF was lowered to 20.8% of its initial value after being subjected to a red LED (50 mW/cm2) for 7 min, indicating very efficient 1O2 production. When the PBS dispersion of the same COF was illuminated with an 808 nm laser of 1.5 W/cm2 intensity, no substantial decrease in DPBF absorbance (92.3%) was found, indicating that an NIR light source would not generate effective 1O2 production. There was no increase in 1O2 generation efficiency when both the red LED and the 808 nm laser were applied, compared to when only the red LED was used. As a result, the red LED was the source of 1O2 generation [In another study, the synthesis of BODIPY-NCOF  was reported via a Schiff base condensation reaction between the free aldehydic group of benzene-1,3,5-tricarbaldehyde and the imine group from tert-butyl(4-aminophenyl) carbamate (NBPDA) neration .Likewise, we discuss more examples on the biomedical applications of COFs, with special emphasis on drug delivery, photodynamic and photothermal therapy, bioimaging, biosensing and biological analysis in The reticular chemistry involves strong bonding to join molecular basic components to form crystalline open structures. It has greatly extended the range of chemical compounds and materials such as organometallic compounds (OMC), metal organic frameworks (MOFs), covalent organic frameworks (COFs), and so on. The linking processes in COFs and MOFs had to be developed to solve the “crystallization challenge”, resulting in crystalline products whose structure could be determined unequivocally using X-ray and electron diffraction analysis tools . The cryIn recent years, the most popular approach to achieve crystalline COFs is to crystallize them via reversible covalent bond formation. Essentially, reversible reaction allows continual bond creation and breaking, and furthermore, defect correction during the construction of the building blocks. Therefore, the system can eventually reach the thermodynamic equilibrium conditions for the final product, whereas in pre-orienting the construction components, the initial or primary step is isolated from the development of strong covalent bonds . Weak cod) is required for 3D growth [The designing principle behind the COF is a condensation reaction. Polymerization processes are centered on the self-condensation of a substituted monomer or the co-condensation of monomer units with two distinct functional groups. In the self-condensation process, single monomers with active functional groups are combined, whereas in the co-condensation reaction, the reaction between distinct monomers with distinct or the same functional groups is combined. To produce a polymer skeleton with an extended 2D structure, covalently connecting monomers requires a planar or linear structure of subunits with symmetry. Essentially, the topologies are aligned to satisfy a topologically directed 2D polymer formation, in contrast to a knot with a 3D branching geometry, i.e., a tetrahedral geometry  or orthogonal (oh) knot in conjunction with C1, C2, C3, C4, and Td organic linker types results in 3D COFs with connections in various combinations to create skeletons and pores [Additionally, the Kagome honeycomb, with an octagonal mesopore in the center and six trigonal micropores on each corner, was formed by linking a Cnd pores . COFs innd pores ,20. In 2nd pores .Apart from this, 3D COFs’ condensation type, topology type and monomer symmetry present in recently reported COFs in the literature are listed in To date, a significant number of synthetic methods have been identified, such as solvothermal, ionothermal, mechanochemical, microwave-assisted, and ultrasonication processes, as given in In the solvothermal synthesis technique, monomers and mixed solvent systems are inserted and degassing is performed by multiple freeze–pump–thaw cycles in a Pyrex tube. After that, the tube is sealed and subsequently heated to a specific temperature for a specific reaction time. COF solvothermal synthesis lasts 3–7 days and demands elevated heating in a sealed tank. In this method, the reaction system’s pressure is critical, since it can have a substantial impact on the COF microstructure and porosity. Likewise, choosing the reaction media and different factors, including the solubility, the rate of reaction, crystal formation, the ‘self-healing’ structure, and the crystal growth rate, is crucial for COF synthesis . FurtherFor example, boronate ester-linked COFs have been synthesized using solvent combinations including dioxane–mesitylene, THF–methanol, and DMAc–o-dichlorobenzene . The fir2, have been explored for COFs synthesis, for example, CTF-1, CTF-2. A thick-walled tube, such as a Pyrex tube, as well as elevated heat (400 °C) and pressure, are the ionothermal conditions necessary for COFs synthesis. In this type of synthesis process, they served as both a solvent and an accelerator. In the last step, ZnCl2 is removed from the resultant CTF mixture and is then cooled to room temperature, crushed, and completely rinsed with water [2, then filtered, washed with water and THF, and finally dried under vacuum. Likewise, ionic liquid as a solvent has provided a convenient, moderate, and green chemistry method for 3D COF synthesis. For example, 3D-IL-COF-1 may be achieved in three minutes, indicating a fast reaction process using ionic liquid. Through this approach, a series of 3D ionic-liquid-comprising COFs has been successfully synthesized. In addition, the ionic liquid was discovered to be reusable, with acceptable activity loss of crystalline materials. Such type of research not only introduces a novel synthesis method for COFs, but also paves the way for green large-scale COF production in industry [In the ionothermal process, ionic liquid/molten salt, i.e., ZnClth water . The powindustry .2 g−1 BET surface area was found in the case of microwave-assisted synthesis techniques, which is somewhat greater than that for the solvothermal synthesized method. Apart from this, microwave solvent extraction methods have many advantages, such as removing copolymers from COFs more efficiently, producing COFs with high permeability, etc. [To counter the limitation of solvothermal synthesis techniques, the microwave assisted synthesis approach has been investigated and reported for the fast production of crystalline porous COFs. Many COFs, i.e., boronate-ester-linked COF-5, 2D COF-5, COF-102, and 3D COF-102, have been reported using microwave-assisted synthesis. Usually, twenty minutes was needed for COF synthesis using microwave irradiation, which is significantly quicker than the 72-h prolonged reaction time necessary under solvothermal synthesis conditions. Furthermore, microwave heating has been shown to improve the properties of COFs, such as the surface area. For example, a 2019 mty, etc. .Since both microwave and solvothermal processes are performed under difficult circumstances , a straightforward synthesis technique is needed for COF synthesis. Mechanochemical synthesis is a synthesis technique which fulfil these needs. Essentially, in mechanochemical synthesis, the monomers are placed into a mortar and crushed with a pestle at ambient temperature to produce COFs. A small quantity of catalyst solution is transferred to the mortar during crushing the subunits, which improves the reagent’s effectiveness. Furthermore, ketoenamine, imine, and hydrazine COFs were synthesized using mechanochemical synthesis ,23. It h2 g−1 [The sonochemical method is another approach to handle the limitations of solvothermal synthesis. Clouds are produced in the solution using ultrasound, which develop and burst in a process known as cavitation, resulting in an extremely high temperature and pressure in the solution, thereby speeding up the rate of the chemical process ,24. For 2 g−1 ,21,22. HMore recently, thin COF films have been reported based on the vapor-assisted COF synthesis method. For example, COF precursors were mixed in appropriate solvent mixture, such as acetone and ethanol, and then drop-casted on a substrate, which was subsequently placed in a desiccator with a reservoir mixture containing a 1:1 ratio of mesitylene and dioxane. A smooth, homogenous COF film was prepared after 72 h at ambient temperature. As a result, the crystallinity of the products was found under strongly influenced solvent reservoirs. The thin film growth was proved on a variety of surfaces, with a variety of COFs, and with the ability to adjust the depth of the layer by altering the quantity of water ,24.−3 Sm−1, owing to its extended conjugated framework, which aids electron transport [Utilizing the Sun’s light/energy has been reported as the most recent synthetic method for COF synthesis. It was observed within three hours of reaction time, light promoted extremely covalently linked and crystalline materials of COFs. For example, COF (hcc-COF) was synthesized using a mixture of hexaketocyclohexane octahydrate (HCH) and 1,2,4,5-benzenetetramine tetrahydrochloride (BTA) (in different solvents) in a quartz tube in an atmosphere of air under simulated visible light of 200–2500 nm wavelength. To ensure effective imine condensation, a small quantity of water and acetic acid were mixed as cocatalysts. In luminous circumstances, it was discovered that COF existed. The synthesized hcc-COF has the greatest electrical properties of 2.22 × 10ransport . Table 2We have found that COFs are promising materials for catalysis, sensing, optoelectronics, small molecules adsorption, gas storage and separation, and drug delivery. This is due to the favorable characteristics of COFs, i.e., an inherent permeability, excellent pore aperture, large surface area, ordered channel structure, low density, high dimensionality, large size, and tunable pore compatibility. In this section, we critically discuss COFs for biological applications using recently reported research.When compared to MOFs, COFs are still in early developmental stages for drug delivery applications; however, it is obvious that the use of COFs for therapeutics is gaining attraction, and significant attempts have been undertaken to develop COF-based therapeutics . The verFurthermore, as compared to PI-COF with a reduced pore diameter, the release rate was lower (60% for PI-COF-4 vs. 49% for PI-COF-5 after 12 h), indicating that pore size and geometry are directly connected to drug delivery in COFs . AdditioTraditional chemotherapy methods suffer from poor efficacy, a high cost, many adverse effects, etc. To overcome all these limitations, phototherapy is a potential treatment method when compared to other cancer therapeutic techniques. It has garnered a lot of interest due to its various advantages, including a high efficacy, low cost, little invasiveness, minimal adverse effects, etc. The use of COF photodynamic and photothermal therapy adds many more advantages, due to their structural and physiochemical features. They result in regular pores with large diameters, allowing large-sized molecules, such as porphyrins and phthalonitriles, to be encapsulated more easily. Moreover, COFs are metal-free analogues of porous materials and should be extremely biocompatible; they might also avoid the possible toxicity of metal containing nanomaterials .1O2 production and a photothermal conversion capacity of 55.9% when exposed to visible light [3O4 nanostructures for PTT, the findings indicated that Fe3O4@COF had a 21.5% photothermal conversion efficiency, which was similar to that of other well-studied photosensitizers, such as Au nanostructures [In recently reported work, nanoscale COFs (NCOF) can be employed as a dual modal (PDT/PTT treatment) platform when synthesized using a simple synthetic method in ambient circumstances. For example, stepwise BDF and guest encapsulation techniques were employed for the successful synthesis of VONc@COF-Por. In this report, PDT and PTT effects were seen separately in covalently grafted porphyrinic PS (Por) and noncovalently loaded naphthalocyanine PTA (VONc). It showed strong le light . Furtherructures . Additioructures . The comAnother porphyrin-containing covalent organic polymer (PCOP) synthesized through a room temperature solution-based ageing technique showed enhanced anticancer effects. The resultant nanoparticles had a high photothermal conversion efficiency (21.7%), as well as a good photodynamic effect. These results indicate that COFs are very promising emerging materials in biological applications. In the future, it is hoped that more functioning COPs will be developed and employed in a variety of biological applications .2S) detection  d3N4 with a high condensation of carbon nitride 2D COF of tri-s-triazine or s-triazine linked via tertiary amines results in a metal-free graphite-like layered structure. It exhibits an appropriate band gap of approximately 2.7 eV and a layer spacing of around 0.33 nm [Furthermore, a graphene-based two-dimensional single-layered nanomaterial has also been reported with good stability, more functionality, and an appropriate band gap for excellent bioselectivity and bioimaging properties. For instance, g-C 0.33 nm .In 2014, researchers used an imine-based COF thin film with a silicon substrate functionalized with an amine for BSA protein and DNA electrochemical detection in aqueous solution . BSA proIn 2021, Mallakpour et al. reported a literature study on the applications of 3D printing technology in water treatment, biomedicine, the electronic industry, and gas removal using metal organic framework (MOF)/covalent organic framework (COF) based materials . Afterwa2 adsorption  monolith for COsorption a, exhibiectivity b. Importsorption .More recently, Bulit et al. reported a new prototype for the synthesis and processing of COFs. This research group convey the combination of microfluidic technologies with 3D printing technology. Therefore, direct-ink writing (DIW) and 3D printing are two other methods that could help COFs handle more information and exhibit more advantages . We hope2, and methane. For example, 3D-COF, i.e., COF-102, has been reported with a high BET surface area of 4650 m2 g−1 and demonstrated a greater CH4 storage capacity of 25 wt% (203 cm3/cm) compared to the 15 wt% capacity of COF-320 at 298 K and 80 bar [−1 H2, 177 mg g−1 CO2, and 11.5 mg g−1 CH4, with stronger selectivity towards carbon dioxide than nitrogen and methane [2/N2 due to the COF’s N2-phobic and CO2-philic properties at 1 bar. Finally, using COF-TpAzo, the storage capacities of 10.6 mg g−1 for H2 and 11.2 mg g−1 for CH4 were also reported [In the present literature, high porosity and crystallinity of COFs have attracted great attention as promising materials for gas storage and separation of essential gases, such as hydrogen, COd 80 bar . Likewisd 80 bar . Further methane . COF-TpAreported .The massive consumption of non-renewable fossil fuels results in serious environmental problems and energy crises. Hence, for promoting a sustainable environment, clean and environmentally friendly energy storage technologies and energy conversion systems are urgently needed to address the global energy demand and environment issues ,62,63,64COFs possess a high crystalline and conjugated properties with a high dimensional geometry. They have been extensively explored in photovoltaic device applications, including dye-sensitized solar cells (DSSCs) and perovskite solar cells (PVSCs). Both 2D and 3D COFs can be used in photovoltaic (PV) applications; however, 3D COFs are less reported in contrast to 2D COFs. Jiang and co-workers reported a chemically stable and photoconductive fullerene-loaded CS-COF, synthesized by the co-condensation of tert-butylpyrene tetraone (PT) and triphenylene hexamine (TPHA), possessing a 0.9% power conversion efficiency with an open circuit voltage of 0.98 V . Later, 2 as a photoelectrode in a dye-sensitized solar cell (DSSC), in order to operate with N719 dye. Under conditions that simulate one sun illumination, an unadulterated DSSC that has not been doped with PI-COFs, has an efficiency of 9.05% in the conversion of electrical power. The short-circuit current density (JSC) is increased by 0.04 weight percent, from 17.43 to 19.03 mA/cm2, and the cell efficiency is increased (9.93%). The increase in JSC is due to the PI-COF’s bifunctionality, which improves charge transfer/injection and inhibits charge recombination through the interaction between PI-COF and N719 dye. PI-COFs also serve as a co-sensitizer and supply a modest amount of photoinduced electrons when exposed to sunshine. The hydrophilicity of PI-COF particles is increased, and the heterogeneous coupling between PI-COF and TiO2 nanoparticles is strengthened, leading to a more effective charge injection when the oxygen plasma’s surface is modified. As a result, an improved JSC of 19.43 mA/cm2 and a power conversion efficiency of 10.46% were observed. This approach to improving the solar efficiency involves doping TiO2 nanoparticles with PI-COFs to modify the photoelectrode, which has the potential to further the development of DSSCs [In another study, polyimide COFs (PI-COFs) were doped in TiOof DSSCs .In this study, two fluorinated side chain conjugated polymers known as P1 (2 F) and P2 (4 F) in tandem polymer solar cells (TPSCs) were investigated. The enhanced absorption coefficients and deep highest occupied molecular orbitals of these conjugated polymers are what cause their high open-circuit voltages (Voc), which are 0.91 and 1.00 V, respectively. P1 or P2 front cells and PTB7-Th rear cells have been effectively combined to create TPSCs using these conjugated polymers. With impressive power conversion efficiencies of 11.42 and 10.05% and high Vocs of 1.64 and 1.72 V, respectively, the improved TPSCs provide excellent power output. By balancing charge mobilities to assess these findings and combining optical and electrical modelling, it is possible to show that all the photovoltaic characteristics in TPSCs have improved simultaneously .Among all energy storage technologies, super-capacitors have drawn attention because of their typical long cyclic stability and high energy density. According to their charge storage mechanism, super-capacitors can be classified as pseudo-capacitors or electrochemical double-layer capacitors. EDLCs can store charge without electron transfer between the electrode and electrolyte interfaces, while in the case of pseudo-capacitors, energy is stored by a reversible, instant intercalation course and a fast-redox reaction with electron transfer between the electrode and electrolyte.−1 [−1 at 0.5 Ag−1 and at a voltage of 0–2.5 V [−1 and a force thickness of 750 Wkg−1. Additionally, it showed brilliant cyclic stability and could hold 88% capacitance after 20,000 charge–discharge cycles.In super-capacitors, charge storage occurs when fast absorption/desorption takes place. The application of COFs in super-capacitors has shown advantageous features, such as (A) a controllable pore structure and high surface area, which can provide room for an electrolyte (in EDLCs); and (B) redox-active functionalities are appropriate for pseudo-capacitive energy storage. Dichter and co-workers were the first who introduced two 2D COFs to super-capacitor applications . Another−1 . In anot−1 . A study 0–2.5 V ,72. In aThe construction and adjustment of photocatalysts exhibiting excellent efficiency, depending on the usual photocatalytic activity, would include factors such as increased visible-light absorption capacity, easier electron–hole splitting, and reduced photo-corrosion over longer periods of time. COFs as a versatile substrate are featured prominently in photocatalysis, and many techniques for increasing their activity based on modified COFs have been devised. The following steps can be taken to improve visible-light absorbance and minimize electrons and hole recombination. The most direct path is to incorporate a relevant functionality to COF. The structural elements from which COF is constructed affect its physiochemical properties, allowing structural alteration of their energy band arrangement. Furthermore, elemental doping can serve as another simple and effective approach to adjust the chemical and physical properties of COFs at the nanoscale. An alternative strategy for improving COFs’ photocatalytic activity is to add either an appropriate chromosphere or a suitable sensitizer. All of the aforementioned methods make use of solar energy throughout a broad spectrum, which is vital for photocatalytic reactions .2 evolution property can be enhanced by substituting ascorbic acid with triethanolamine (TEOA) as an electron donor, though with the compromise of a faster deactivation of the COF. With the addition of 10 vol% TEOA, the H2 evolution rate was found to be 1970 μmol h−1 g−1 corresponding to a quantum efficiency of 2.2%. This rate was found to be enhanced approximately three times when compared with standard photocatalytic systems, such as Pt-modified amorphous melon, crystalline poly (triazine imide), and other carbon nitrides, [−1 g−1, which is about 10% larger than that of the N3-COF. COF-based photocatalysts for hydrogen production have also been produced by modifying the fundamental components and connections. For example, by expanding alkynes with the alteration of peripheral heteroaromatic building blocks, a series of planar pyrene-based COF (A-TEXPY-COFs) was synthesized. In this system, the most advanced donor has the highest H2 evolution rate of 98 mmol h−1 g−1 [Fuel scarcity is currently one of the hardest challenges to address, especially in a safe and renewable manner. Hydrogen evolution by UV illumination is the foremost important renewable energy source. In 2014, the very first application of a COF for photocatalytic hydrogen generation was discovered. Due to the high electrical conductivity and electron-withdrawing properties, the triazine-based structure showed high performance for hydrogen evolved reactions. For instance, coupling 2,5-diethoxy terephthalohydrazide (DETH) with 1,3,5-tris-(4-formyl-phenyl) triazine (TFPT) gives crystalline hydrazone-linked COF (TFPT-COF). The pore size of TFPT-COF was found to be 3.8 nm, which the largest among all the hydrazone linked COFs. Furthermore, the Hitrides, ,74,75. I h−1 g−1 .2 production research, as the breakage of the O–H bond, the creation of the O–O bond, and the high overpotential with slow O–O formation kinetics are all involved in water oxidation in the case of oxygen evolution, which is more complex, with the rate-determining step (four-electron redox process) in total water splitting. Recently, photocatalytic water separation has been discovered using covalent triazine-based frameworks (CTFs), a class of semi-conductive materials. Their tunable band gap and easy fabrication provide them with many possibilities for research and development. CTF-1, for example, was produced via microwave-assisted condensation at various powers and was subsequently used in water splitting. Under similar photocatalytic circumstances, the material (CTF-0-M2), produced using the microwave technique, has an excellent photocatalytic activity for H2 production (up to 7010 mol h−1 g−1). This is about seven times consistently higher than the other framework (CTF-0-I) created using traditional ionothermal trimerization. After seven cycles in visible light, this results in a higher turnover number (TON) of 726 for the Pt cocatalyst. The material produced via the ionothermal technique, on the other hand, is the most efficient in releasing oxygen. Under similar experimental circumstances, CTF-0-I initially generates approximately six times more oxygen gas than CTF-0-M2. The quantum efficiency of CTF-0-I is evident. CTF-0-I produces oxygen with an obvious quantum efficiency (AQY) of 5.2% at 420 nm in the absence of a cocatalyst [Photocatalytic water oxidation research with COF photocatalysts is considerably less developed than photocatalytic Hcatalyst .2) emissions, causing significant environmental issues such as the greenhouse effect, temperature increase, climate change etc. Photocatalytic CO2 reduction to clean renewable resources has raised attention in recent years as a promising approach for simultaneously tackling environmental and energy concerns. COFs, the potential photocatalytic candidates, are acknowledged as a spectacular platform for photocatalytic CO2 reduction due to their strong CO2 absorption capacity and specificity.With the world’s population and economy expanding at a rapid pace, increased coal and oil use has resulted in increased carbon dioxide  and N3COF (1412 m2 g−1) in this work, along with numerous accessible nitrogen sites, resulted in strong CO2 absorption, facilitating the photocatalytic reduction of carbon dioxide to methanol. The total quantity of CH3OH produced over N3-COF after 24 h of visible light irradiation was 13.7 mmol g−1, which was more than ACOF-1 (8.6 mmol g−1). In comparison to ACOF-1, N3-COF, with electron-poor triazine groups, can maintain the negative charge produced on the COF. This is critical for improved photocatalytic activity [Azine-based COFs containing p-stacking aromatic units are considered as the most promising photocatalytic possibilities. The separation and transmission of photo-induced electrons/holes might be aided by a large, conjugated structure. Two azine-linked crystalline COFs, such as ACOF-1  and Nactivity ,78,79.This review has discussed the significant developments in biomedical applications using COFs, due to their numerous advantages, such as a high surface area, a porous structure, exceptional modification ability, etc. However, despite such desirable attributes, COFs still face many issues in being applied for successful practical biomedical applications, i.e., hydrolytic stability, targeted delivery, biocompatibility, challenges in synthesis techniques, and so on. In a real sense, they tend to decline the success rate for obtaining fruitful results. As a result, it becomes significantly important to address the issues via extensive research to overcome the limitations of COFs as far as biomedical purposes are concerned. Furthermore, the integration of functional groups to the synthesized COFs enhances their biological applications. However, tagging functional groups on the surface of COFs is still challenging due to poor stability and other unfavorable conditions.Secondly, there is an urgent need for novel synthesis strategies to produce large numbers and quantities of COFs for COF technology in the near future. Because most COF synthesis processes involve condensation reactions between the same or different organic ligands in a suitable solvent ratio, this hinders large-scale COF manufacture. To overcome this limitation, room-temperature and solvent-free synthesis processes must be developed for the large-scale manufacturing of COFs. Moreover, understanding the relevance of reversible bond formation has been vital in the development of novel COFs and enhancing their crystallinity, although determining their structure remains a challenge. The structure and many other key characteristics of COFs, such as bond connectivity, the nature of bonds, the position of atoms, and the location of guest molecules remains inaccessible. Recently, several researchers succeeded in synthesizing large crystals of COFs by a slow crystallization process. This enables us to understand the COFs’ structure in a more detailed manner via single-crystal X-ray diffraction analysis.COFs exhibit unique properties that allow them to be used in a variety of fields, such as catalysis, energy storage, gas storage and separation, optoelectronics, and so on. Their advancements offer more opportunities for researchers in the near future. When compared to other porous materials, COFs offer several unique applications. Post-synthetic modifications further improve the conductivity and electrocatalytic activity of COFs. Importantly, COFs can also be used as a precursor in the development of carbon nanomaterials containing a single metal or metal clusters, which can offer excellent electrocatalytic performance. The cost issues and synthetic challenges, such as morphology and size control, discourage COFs materials from being used as precursors in making carbon materials; however, using COFs as precursors to prepare single-atom catalysts (SACs) could be an interesting path to pursue. When considering industrial applications, the cost is a crucial concern; therefore, by making COF materials scalable and by achieving functional criteria, such as long span stability, reversibility, processability, and high performance.Moreover, most of the research on COF involves imine, boroxine, hydrazine, and cyanide linkages. Very few COF synthesis reactions contains peptide linkages (condensation between acid and imine bonds). Efforts must be made by researchers to synthesize COFs based on acid–base reactions. Hopefully, COFs will gain a foothold in the industrial field soon.


Caenorhabditis elegans FAMP-1 (formerly named M70.4), which has been recognized as a tentative ortholog of mammalian FAM3 members or protein-O-mannose β-1,2-N-acetylglucosaminyltransferase 1 (POMGnT1). Missense mutations in the N-terminal domain, named Fam3L2, caused defects in memory-based thermotaxis but not in chemotaxis behaviors; these defects could be restored by AFD neuron-specific exogenous expression of a polypeptide corresponding to the Fam3L2 domain but not that corresponding to the Fam3L1. Moreover, human FAM3C could also rescue defective thermotaxis behavior in famp-1 mutant worms. An in vitro assay revealed that the Fam3L2 and FAM3C can bind with carbohydrates, similar to the stem domain of POMGnT1. The athermotactic mutations in the Fam3L2 domain caused a partial loss-of-function of FAMP-1, whereas the C-terminal truncation mutations led to more severe neural dysfunction that reduced locomotor activity. Overall, we show that the Fam3L2 domain-dependent function of FAMP-1 in AFD neurons is required for the thermotaxis migration of C. elegans and that human FAM3C can act as a substitute for the Fam3L2 domain in thermotaxis behaviors.The family with sequence similarity 3 (FAM3) superfamily represents a distinct class of signaling molecules that share a characteristic structural feature. Mammalian FAM3 member C (FAM3C) is abundantly expressed in neuronal cells and released from the synaptic vesicle to the extracellular milieu in an activity-dependent manner. However, the neural function of FAM3C has yet to be fully clarified. We found that the protein sequence of human FAM3C is similar to that of the N-terminal tandem domains of  Significance StatementCaenorhabditis elegans FAMP-1; however, functional similarity remains to be clarified. We found that FAMP-1 is required for the memory-based thermotaxis behaviors of C. elegans, and missense mutations in the N-terminal Fam3L2 domain of FAMP-1 resulted in defective thermophilic migration. Unexpectedly, exogenous expression of human FAM3C as well as the Fam3L2 domain in AFD thermosensory neurons rescued the defects in thermophilic migration in famp-1 mutants. Additionally, similar to the stem domain of POMGnT1, the Fam3L2 domain and FAM3C bound to GlcNAc but the thermotaxis-defective Fam3L2 mutants did not. Our findings suggest that these proteins share a common mechanism underlying the functions observed in neuronal cells.Human FAM3C shows sequence homology to the stem domain of protein-O-mannose β-1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) and the N-terminal domains of N-acetylglucosamine (GlcNAc) residue to O-mannose in glycoproteins. Homozygous mutations of POMGnT1 lead to defects in the synthesis of O-mannosyl glycan and cause muscle–eye–brain disease characterized by congenital muscular dystrophy, ocular abnormalities, and brain malformation with mental retardation (The family with sequence similarity 3 (FAM3) superfamily, composed of FAM3A, FAM3B, FAM3C, and FAM3D, was predicted to share a classical cytokine-like four-helix bundle structure in an earlier study . Howeverardation .FAM3C shows ubiquitous expression under physiological conditions and plays a role in hepatic gluconeogenesis repression and osteoblast differentiation in mice , 7. In a0 phase of the cell cycle . Missense mutations in the N-terminal domain of FAMP-1 caused defects in thermotaxis but not in chemotaxis behaviors; these defects could be restored by the AFD neuron-selective expression of this domain. Intriguingly, human FAM3C could also rescue the thermotaxis behavior of famp-1 mutants. Moreover, an in vitro assay revealed that this domain and FAM3C can bind with carbohydrates, similar to the stem domain of POMGnT1. These results suggest that FAMP-1 is a functional ortholog of mammalian FAM3C.In our previous studies, we have found that FAM3C is widely expressed in neuronal cells of the mammalian brain, including cerebrocortical pyramidal neurons that are fully differentiated and reside in the Gll cycle . Neuronall cycle , 19. Secll cycle . Althougll cycle . Hence, ehaviors . In thisC. elegans canonical orthologs of FAM3 superfamily members. However, human FAM3 genes had significant sequence similarity to two coding regions of famp-1/m70.4 (WBGene00019786) that encode N-terminal tandem domains, here referred to as FAM3-like domains 1 and 2 (Fam3L1 and Fam3L2), respectively : VC40639 famp-1(gk737833), VC40642 famp-1(gk739534), VC40848 famp-1(gk845538), VC20615 famp-1(gk355956), VC40532 famp-1(gk681625), VC40952 famp-1(gk884912), VC40352 famp-1(gk589965), VC20300 famp-1(gk195774), VC40447 famp-1(gk642110), VC41013 famp-1(gk927637), and VC20156 famp-1(gk314930) strains, harboring S136F, S197R, G210R, P291S, G310E, M312I, T362I, N427I, and H443Y missense and R400X and W445X nonsense famp-1 gene mutations, respectively . All mutants showed normal gross morphology. A locomotory rate assay revealed that all mutants, except the N427I mutant and two nonsense mutants, exhibited equivalent locomotor activity to wild-type worms  were exclusively localized in the Fam3L2 domain , 2722, 2ion Fig.  and D. Tnt worms . Thus, tfamp-1 in AFD sensory neurons was revealed in a previous study employing single-cell RNA-sequencing analysis  mutant  mutant exhibited a reduced head avoidance reaction, as previously reported  mutant  also shoreported .The reversal frequency of athermotactic mutants and wild-type N2 worms did not differ significantly . These fC. elegans migration behavior, we also performed chemotaxis assays of the famp-1 mutants as previously described (https://www.cengen.org), which shows that famp-1 is not expressed in the AWC and AWA chemosensory neurons.Given that chemotaxis is another type of escribed . Wild-tyescribed . Therefofamp-1 is expressed in multiple types of neurons and could be involved in various behaviors such as egg-laying and escape from higher cultivation temperature. The egg-laying muscles receive synaptic input from two classes of neurons, namely the HSN and VC, whereas non-neuronal cells, e.g. vulval muscles, uv1 gland cells, and VulD epithelial cells, are also involved in egg-laying  neuronal FAMP-1 is required for the thermotaxis behaviors of C. elegans; (3) missense mutations in the Fam3L2 domain of FAMP-1 cause defects in thermophilic navigation; (4) exogenous expression of the Fam3L2 domain in AFD thermosensory neurons can rescue the athermotactic defect of famp-1 mutants; (5) Fam3L1 is not functionally redundant with Fam3L2 in thermotaxis behaviors; (6) human FAM3C can substitute for Fam3L2 in restoring the thermotaxis of famp-1 mutants; and (7) FAM3C and the Fam3L2 domain but not the Fam3L1 and athermotactic mutant Fam3L2 domains bind to GlcNAc. These findings suggest that the Fam3L2 domain-dependent function of FAMP-1 in AFD neurons, which has yet to be clarified, is nonetheless required for the thermophilic migration of C. elegans. They also indicate that human FAM3C/A can rescue the loss-of-function of the Fam3L2 domain in thermotaxis behaviors.Our results indicate the following: (1) sequence similarity exists among FAM3 superfamily proteins, the Fam3L domains of 2+ into AFD neurons (2+- and diacylglycerol-activated protein kinase C)-depletion resulted in athermotactic behaviors after precultivation at 20°C  or Fam3L2 mutants, suggesting that Fam3L2 mutations perturbed thermal memory formation rather than thermal sensation in AFD neurons.The disruption of AFD functions caused multiple behavioral defects, including cultivation temperature-independent athermotactic navigation , a robusfamp-1 mutants. Fam3L2 shares 34% and 30% similarity with human FAM3C and the stem domain of POMGnT1, respectively .The orthologous relationship among FAMP-1, POMGnT1, and FAM3 members remains obscure. Many nematode strains have single or duplicated FAM3-like domains, which are always followed by a domain with homology to a part of the C-terminal catalytic domain of POMGnT1 . Howeverdiverged  52, 53), 53C. elC. elegans, which could be rescued by exogenous expression of human FAM3C as well as the Fam3L2 domain in AFD thermosensory neurons. An in vitro assay indicated that the binding of FAM3C and the Fam3L2 domain to GlcNAc was required for this restoration. Our findings suggest functional similarity of these proteins, sharing a common molecular mechanism mediated through carbohydrate-binding.Several missense mutations in the Fam3L2 domain of FAMP-1 precluded the memory-based thermotaxis behaviors of C. elegans Bristol N2 strain) were cultivated at 20°C on nematode growth medium plates seeded with Escherichia coli OP50-1. The famp-1 mutants, derived from ethyl methanesulfonate mutagenesis, were obtained from CGC (University of Minnesota) and repeatedly backcrossed to N2 in an attempt to create each isogenic mutant strain. Germline transformation was performed by coinjecting experimental DNA (2 ng/µL) and the coinjection marker pKDK66  into the gonad of adult worms , or gcy-18p:: human FAM3A (codon optimized) were obtained from M312I and H443Y mutant worms. Each genotype was confirmed using PCR with specific primer pairs. The unc-14, gcy-18, ttx-3, and glr-3 promoters drove pan-neuronal, AFD neuron-specific, AIY neuron-specific, and RIA neuron-specific expression, respectively  mutant strains were also obtained from CGC.Wild-type worms  was placed on aluminum plates attached to a Peltier thermocontroller. The linear thermal gradient was confirmed using a thermal camera , and approximately 50 to 200 worms were placed at the 20°C region of the gradient. After 60 min, the worms were killed with chloroform gas. The agar surface of the assay plates was divided into eight subregions along the gradient with scores of 1 to 8, and the thermotaxis index was calculated as follows:iS is the number of worms in each subregion and totalN is the total number of worms on an assay plate.To conduct a population thermotaxis assay, a nematode growth medium plate  containing 10 mL of thermotaxis medium . Images were captured with a CCD camera DP73 (Olympus).PY1322 18 worms , which eWorms were exposed to noxious heat using an electronically heated metal tip. The temperature at 3.0 mm from the tip of a platinum wire (0.5 mm in diameter) was 33.0°C ± 1.0°C. The assays were performed in a room with a constant temperature of 20°C and a humidity of at least 60%. The heat stimulus was presented in front of the worm, and the initial response was classified according to a previous study  as folloAdult worms were washed twice, after which approximately 100 to 200 worms were placed on a 6-cm agar plate with food and cultured at 20°C for 60 min. The left half of the surface of the assay plate was then heated to a noxious temperature at 29, 31, or 33°C using a Peltier thermocontroller. Before and 60 min after heating, the number of worms on the right and left surface was counted. Assays were repeated three times for each genotype.The frequencies of body bends and spontaneous reversal behavior were evaluated according to the methods in a previous study . Well-feE. coli OP50-1. The number of laid eggs per worm per hour was counted using a standard dissection stereomicroscope.Worms were picked at the early L4 stage and cultured at 20°C before experiments. During the experiment, the worms were allowed to lay eggs for 3 h on a nematode growth medium plate seeded with Total RNA was extracted from wild-type N2 and M312I-mutant worms using TRIzol reagent (Invitrogen). The 1st strand DNA was synthetized using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO). Quantitative PCR was performed on a Light Cycler 480 System II (Roche Diagnostics) using KOD SYBR qPCR Mix (TOYOBO). The following PCR primer pairs were used: Ce-hsf-F and Ce-hsf-R for hsf-1: 5′-CGAAAGATGACTCCACTGTCC-3′ and: 5′-GTCCTCCACAGTTCTTGCC-3′, respectively; Ce-ama-F and Ce-ama-R for ama-1: 5′-TGGAACTCTGGAGTCACACC-3′ and 5′-CATCCTCCTTCATTGAACGG-3′, respectively. The ama-1 mRNA served as an internal control.HEK293 cells were transfected with expression plasmids for Myc-FLAG-tagged human POMGnT1, Myc-FLAG-tagged human FAM3A, 3xFLAG-tagged human FAM3C, FLAG-tagged Fam3L1, and FLAG-tagged Fam3L2 using Lipofectamine LTX (Invitrogen). Extracts of transfected cells were mixed with fucose-, mannose-, lactose-, GlcNAc-, or GalNAc-conjugated resins  and incubated at 4°C overnight. Carbohydrate-conjugated resin-bound proteins were eluted using a buffer containing 0.1 M of each free carbohydrate. The eluates were subjected to immunoblotting, and the blots were validated with anti-FLAG antibody (Sigma-Aldrich).t-test for two-group comparisons and one-way ANOVA followed by Tukey’s post hoc test for three (or more)-group comparisons. Statistical significance was defined at *P < 0.05 or **P < 0.01.Data are presented as means ± SEM. Statistical analysis was performed using the two-tailed unpaired Student’s pgac242_Supplemental_FileClick here for additional data file.


RhoGAP proteins are key regulators of Rho family GTPases and influence a variety of cellular processes, including cell migration, adhesion, and cytokinesis. These GTPase activating proteins (GAPs) downregulate Rho signaling by binding and enhancing the intrinsic GTPase activity of Rho proteins. Deleted in liver cancer 1 (DLC1) is a tumor suppressor and ubiquitously expressed RhoGAP protein; its activity is regulated in part by binding p120RasGAP, a GAP protein for the Ras GTPases. In this study, we report the co-crystal structure of the p120RasGAP SH3 domain bound directly to DLC1 RhoGAP, at a site partially overlapping the RhoA binding site and impinging on the catalytic arginine finger. We demonstrate biochemically that mutation of this interface relieves inhibition of RhoGAP activity by the SH3 domain. These results reveal the mechanism for inhibition of DLC1 RhoGAP activity by p120RasGAP and demonstrate the molecular basis for direct SH3 domain modulation of GAP activity. Inhibition of DLC1 RhoGAP by p120RasGAP allows for crosstalk between the Rho and Ras pathways. Here, Chau et al. present the co-crystal structure of DLC1 RhoGAP domain and p120RasGAP SH3 domain and probe the interaction with biochemical studies. The Rho (Ras homolog) subfamily of small GTPases, including the prominent members RhoA, Cdc42, and Rac1, function in actin cytoskeleton dynamics to affect such cellular processes as cell migration, adhesion, and morphology. Additionally, they act in cell cycle progression, vesicular trafficking, and endocytosis among other functions2.The Ras superfamily of small GTPases are molecular switches that cycle between GTP- and GDP-bound states in response to stimuli. These nucleotide-binding states reflect the “on” and “off” signaling conformations of these proteins, respectively. The GTPase cycle is modulated by the guanine nucleotide exchange factors (GEFs), which facilitate the exchange of GDP for GTP to activate GTPase signaling, and the GTPase activating proteins (GAPs), which enhance the intrinsic GTP hydrolysis activity of the GTPases and downregulate GTPase signaling4). RhoGAPs enhance the intrinsically inefficient catalytic activity by directly binding the GTP-bound GTPase and contributing a conserved arginine residue, the so-called catalytic “arginine finger”, to stabilize the transition state of the GTPase enzyme active site and catalyze the reaction6. The human genome encodes nearly 70 RhoGAPs, including p50RhoGAP, the p190RhoGAP proteins, and the Graf, ARAP, chimaerin, and DLC proteins7. Many of these RhoGAPs are multidomain proteins, and these domains are implicated in regulation of GAP activity as well as scaffolding functions for crosstalk with other signaling pathways8. Though some RhoGAPs are suggested to be regulated by mechanisms including localization and post-translational modification, much remains to be discovered at the molecular level regarding how their RhoGAP activities are controlled4.Activation of Rho GTPase signaling is facilitated by Rho-family specific RhoGEFs, including the Dbl-homology (DH) domain-containing proteins such as SOS and BCR, while downregulation of Rho GTPase signaling is provided by specific RhoGAP proteins. In addition, Rho GDP dissociation inhibitors (GDIs) also downregulate Rho activity by preventing exchange of nucleotide and membrane localization of the GTPases  encodes a ubiquitously expressed Rho GTPase-activating protein that is a tumor suppressor and can regulate RhoA activity both in vitro and in vivo10. DLC1 is the prototypical member of a family that in mammals includes the related genes DLC2 (STARD13) and DLC3 (STARD8), but in worm and fly is conserved as a single DLC gene14. It was first identified as a gene deleted in multiple primary hepatocellular carcinoma (HCC) tumors and liver cancer cell lines15 and upon cloning was observed to be homologous to the rat p122RhoGAP16. Deletion of DLC1 is embryonic lethal, and thus its critical role in cytoskeletal organization was established17. DLC1 has been shown to act on Rho family GTPases RhoA, RhoB, and RhoC, and to a lesser extent on Cdc42, Rac1 and TC109. In addition to the RhoGAP domain located in their central region, each of the DLC proteins contain a sterile alpha motif (SAM) domain in their N-terminus, and a steroidogenic acute regulatory-related lipid transfer (START) domain near their C-terminus . A serine-rich linker region (LR) between the SAM and RhoGAP domains is also present and includes a focal adhesion targeting (FAT) region. Localization of DLC proteins and regulation of the RhoGAP domain are thought to be mediated by the accessory domains18, and by partner proteins, including tensin proteins, focal adhesion kinase and talin24. However, the molecular mechanisms of regulation and targeting remain poorly understood.The nus Fig. 11,15. A RASA1; RasGAP, p120GAP). This was the first RasGAP identified and is a key regulator of Ras signaling28. p120RasGAP is a multidomain protein containing two Src homology-2 (SH2), one Src-homology-3 domain (SH3), a Pleckstrin homology (PH) domain, presumptive calcium-binding C2 domain, and a RasGAP domain  motifs31. Instead, the SH3 domain was first observed by yeast two hybrid to bind the DLC1 RhoGAP domain and suppress its RhoGAP activity32, but the molecular mechanism was not determined33.One major binder and regulator of DLC1 is the RasGAP protein, p120RasGAP . Its closest structural neighbor is the GAP domain of p50RhoGAP , which is of variable length, sequence and structure in the RhoGAP family7, is partially disordered in this structure  shows only minor conformational changes , potentially due to crystal packing and distal from the SH3 binding site  adopts a conserved overall structure that is highly similar to the GAP domain of other RhoGAP proteins, including the previously determined structure of the isolated DLC1 RhoGAP domain . Several previous structure determinations of the apo form of p120RasGAP SH3 domain have been reported: PDB IDs: 2J05 and 2J0643, 4FSS , 2GQI , and 2M51 . The structure here superposes well with these previous structures with RMSD values ranging from 0.4 to 0.8 Å over 58 equivalent Cα positions  adopts a beta sandwich fold comprised of five strands (β1-β5) that form a β-barrel. The strands are connected by loops termed the RT loop (β1-β2), n-Src loop (β2-β3), distal loop (β3-β4), and a 3/10 helix (β4-β5) . Because the RhoGAP domain of DLC1 is highly similar to other RhoGAPs , we can compare the SH3 binding site with the predicted interface between DLC1 and RhoA: the RhoA binding site on DLC1 is predicted to be highly similar to that of p190RhoGAP  conformation of Rho GTPases at a binding site that includes the switch I and switch II regionsion Fig. . Notablyion Fig. .46. Similar to assays presented for Ras47, this method has the advantage of directly monitoring the production of inorganic phosphate by hydrolysis without the need for a fluorescently tagged GTP analog. This assay also avoids any steric interference by a fluorescent moiety. DLC1 displays preference for RhoA, RhoB and RhoC over the other Rho GTPase family members7; therefore, to assess GAP activity we assessed DLC1 activity on RhoA hydrolysis. Additionally, to allow for nucleotide exchange and RhoA to perform multiple hydrolysis cycles we performed these assays in the presence of both EDTA and magnesium.To validate the molecular basis of p120RasGAP inhibition of DLC1 we conducted biochemical GTPase activity assays. We established an in vitro RhoA GTPase activity assay that utilizes the malachite green colorimetric reagent to directly monitor production of inorganic phosphate50 of 0.6 μM   of 0.6 μM measured by isothermal titration calorimetry and the Ki of 0.6 μM determined by kinetics33.Our initial assays demonstrated that the inefficient intrinsic GTP hydrolysis by purified RhoA is catalytically enhanced in the presence of increasing concentrations of DLC1 RhoGAP domain Fig. . In contM) Table . Althoug50 values 4-fold and over 30-fold lower for the Y290A and E298A mutants, respectively  are comprised of aromatic residues in the RT loop, β4, and 3/10 loop and are conserved in most PxxP-binding SH3 domains  polyproline II (PPII) helical peptidesins Fig. a–c.Fig. 43, and our co-crystal structure confirms this divergence. These structures suggest that p120RasGAP may not bind canonical polyproline-containing ligands since Leu-288 and Leu-335 replace aromatic residues in xP1, and Val-332 replaces a typical Proline in xP2  domain autoinhibits the RhoGAP activity by binding and masking the RhoGAP active site67. Similarly, p50RhoGAP is thought to be autoinhibited by binding of its BCH domain to the RhoGAP active site68. However, molecular level details of these interactions are currently unobserved. Additionally, RhoGAPs may be inhibited in trans by a binding partner, like DLC1 by p120RasGAP, but the details of regulation are unknown. Our structure, therefore, provides a molecular level observation of inhibition of a RhoGAP domain by direct masking of the catalytic arginine finger. Whether other RhoGAP proteins are regulated by masking of the arginine finger by direct protein binding remains to be revealed.Many RhoGAP proteins are multidomain proteins, and regulation of their RhoGAP activities may involve intramolecular inhibition via direct interaction 70), or as observed for the p67phox SH3 binding p47phox via both polyproline and a unique helix-turn-helix structure that binds the specificity site , and this regulation mechanism may be shared among other SH3-domain-containing GEFs like collybistin I and intersectin-1. Thus, the SH3 domain fold may play an expanded role in the regulation of Rho family GTPase pathways.It remains to be seen if other SH3 domains can directly regulate RhoGAP activity, but a previous study suggested that the SH3 domains of Src, Crk, and Nck do not act as inhibitors of DLC1 activity77), while correctly predicting the overall fold have not adequately predicted the fine details of the structure. Alphafold presents helix αG in a position that is slightly extended away from the RhoGAP core, similar to other RhoGAP structures. In contrast, both experimentally determined crystal structures of DLC1 RhoGAP domain ) reveal that this helix is packed closer against the RhoGAP domain. This compaction allows interaction with the SH3 domain, whereas the protruded position it creates steric clashes with the SH3 domain.  was a gift from Cheryl Arrowsmith . cDNA encoding the SH3 domain of human p120RasGAP  was inserted into pGEX-6p1 vector (Cytiva) for expression as a GST-fusion protein, and the SH2-SH3-SH2 domains (residues 172–443) or RasGAP domain (residues 714–1047) were inserted into a modified pET vector containing a His6-tag and TEV cleavage site. Wild-type human RhoA cDNA (Uniprot ID: P61586) was inserted into pGEX-6p1 (Cytiva) for expression as a GST-tagged protein. A stop codon was introduced after residue 181 by Quikchange Mutagenesis (Agilent) to encode a truncated protein (residues 1–181) lacking a portion of the C-terminal hypervariable region to avoid modification and membrane localization. Site directed mutagenesis of SH3  and DLC1  was also performed with QuikChange Mutagenesis (Agilent). Human H-Ras cDNA encoding residues 1–167 (UniProt ID: P01112) was subcloned into a modified pET vector for expression as N-terminally His6-tagged protein. All recombinant protein expression was performed in BL21(DE3) or Rosetta(DE3) E. coli cultures by induction with 0.2 mM isopropyl β-d-thiogalactopyranoside at cell density OD600 = 0.6–0.8 for 18 h at 18 °C and harvested by centrifugation.A pET28-MHL expression plasmid for the His6-DLC1, His6-H-Ras, His6-p120RasGAP SH2-SH3-SH2 or GAP domain protein were resuspended in lysis buffer containing 50 mM HEPES pH 7.3, 500 mM NaCl and 20 mM imidazole, and cells expressing GST-SH3 or GST-RhoA were resuspended in lysis buffer containing 20 mM Tris pH 7.4, 150 mM NaCl and 1 mM dithiothreitol. Cells were lysed by three freeze/thaw cycles in the presence of 0.1 mg/ml lysozyme followed by sonication . Total cellular lysate was clarified by centrifugation at 5000 × g at 4 °C for 1 h. His6-tagged DLC1 RhoGAP, HRas, p120RasGAP SH2-SH3-SH2 or GAP domain proteins were purified by applying clarified lysate to nickel-nitrilotriacetic acid (Ni-NTA) agarose beads (Qiagen) for 1 h, which were then washed with lysis buffer prior to step-wise protein elution with lysis buffer containing increasing concentrations of imidazole (40 mM–100 mM–250 mM–500 mM). Fractions containing protein of interest were pooled and applied to size exclusion chromatography (Superdex75 Increase) column equilibrated in buffer containing 20 mM Tris pH 8.0, 150 mM NaCl. The final protein was concentrated in a spin concentrator . H-Ras and p120RasGAP GAP domain proteins were additionally purified by anion exchange chromatography (Mono Q 5/50 GL Cytiva) in 20 mM Tris pH 8 and eluted by a gradient of NaCl from 0.05 to 1 M. DLC1 RhoGAP was used in its His6-tagged form for both crystallization and biochemical assays; likewise, His6-HRas and His6-p120RasGAP GAP domain were used in biochemical assays.Cells expressing HisGST-SH3 or GST-RhoA proteins were purified by affinity chromatography by incubating clarified lysate with Glutathione Sepharose beads (Cytiva) in a gravity flow column at 4 °C for 2 h. Beads were washed with 20 column volumes of lysis buffer, and resuspended in lysis buffer at a 1:1 bead:buffer ratio by volume. GST-fusion proteins were proteolytically cleaved by addition of GST-Prescision protease on-bead. Tag-free SH3 or RhoA proteins were collected as flow-through and further purified over size exclusion chromatography  in buffer containing 20 mM Tris pH 8.0 and 150 mM NaCl. Proteins were concentrated in spin concentrators .6-DLC1 RhoGAP and GST-p120RasGAP SH3 were mixed prior to lysis, which was carried out as described above. The complex, which formed in solution, was purified over glutathione affinity chromatography (Cytiva), liberated from beads by PreScission protease cleavage of the GST tag, and purified by size exclusion chromatography in 20 mM Tris pH 8.0, 150 mM NaCl to yield the protein complex and efficiently separate the excess p120RasGAP SH3 alone.For co-crystallization protein samples, cells expressing HisCrystallization trials were carried out with a protein sample mixture consisting of a copurified complex of DLC1 RhoGAP with p120RasGAP SH3 at 18 mg/ml. Screen setup was performed using a TTP Labtech Mosquito in sitting-drop vapor diffusion setups at ambient temperature (∼22 °C). Initial co-crystals were obtained in PEG/Ion Screen (Hampton Research) condition B4, comprised of 0.2 M Magnesium nitrate, 20% PEG 3350, pH 5.9. Optimal crystals, which grew as rods with approximate dimension 100 μm × 20 μm × 20 μm, were obtained in 0.2 M Magnesium nitrate, 16.5% PEG 3350, in 4 μl hanging drops (2 μl of protein complex plus 2 μl reservoir solution) over 1 ml reservoir solution. Crystals were harvested and cryopreserved in reservoir solution supplemented with 20% ethylene glycol and flash cooled in liquid nitrogen.78 to 3.2 Å resolution in spacegroup H3 with unit cell dimensions a = b = 143.7 Å, c = 152.8 Å, α = β = 90°, γ = 120°. XTriage analysis predicts four copies of the 1:1 complex per asymmetric unit with a solvent content of 46.5%.X-ray diffraction data were collected from a single crystal at the Northeastern Collaborative Access Team (NECAT) Beamline 24-ID-E at Argonne National Laboratory Advanced Photon Source. X-ray data were processed and scaled in HKL200079. Four copies of the DLC1 RhoGAP domain were placed using the previously unpublished DLC1A structure (PDB ID: 3KUQ39) as a search model, with final TFZ score of 34.5 and LLG 1831. Next, placement of two of four expected SH3 domains was achieved using the crystal structure of p120RasGAPSH3 domain (PDB ID: 2J0543) as search model with TFZ scores of 18.0 and 20.5; placement of the third and fourth copies failed. Each SH3 domain formed an identical interface with an individual DLC1RhoGAP copy in the asymmetric unit. Thus, the final two copies of SH3 were placed manually based on this interface with the remaining two DLC1RhoGAP copies and validated by refinement. Phenix Autobuild80, and manual building in Coot81, were performed. Refinement was carried out in Phenix82. A total of 1010 protein residues were modeled, with 5 N-terminal vector-derived residues in each DLC1 protein chain. All crystallography software was compiled by SBGrid83. Raw diffraction images have been deposited to the SBGRID Data Bank: 10.15785/SBGRID/876. Protein model coordinates and structure factors have been deposited to the Protein Data Bank with accession number 7TPB and prepared using PDB Extract version 3.27. Crystallization figures were generated in ccp4mg84. Protein interface calculations were performed on the PISA server  at the European Bioinformatics Institute44, and structure-based alignments were run on the Dali server85.The structure was solved by molecular replacement using Phenix Phaser2 and 2 mM Ethylenediaminetetraacetic acid in final reaction volume of 80 μl. The reactions were initiated by addition of GTP at 50 μM. To monitor the effect of p120RasGAP SH3 domain, DLC1 RhoGAP protein at 0.05 μM and a titration series from 0 to 20 μM of wild-type or mutant SH3 domain protein were included. To assess the activity of wild-type and mutant DLC1, 0.1 μM DLC1 proteins were mixed with 0, 1, or 10 μM SH3 domain protein. The reactions were carried out at room temperature for 40 min in clear bottom 96-well plates (Corning) and terminated by addition of 20 μl of the malachite green working reagent, which was prepared as per the manufacturer’s instructions (Sigma Millipore), followed by color development for 30 min. A single absorbance reading at 620 nm for each sample was measured at room temperature in a Synergy H1 plate reader (BioTek) using Gen5 3.11 software. A standard phosphate curve with concentrations from 4 to 40 μM was used to generate a linear standard curve and was used to calculate the phosphate generated in each sample well. Reactions in the absence of added GTP were included to control for background phosphate content signal, which was found to be negligible. The IC50 of SH3 domain or SH2-SH3-SH2 domain inhibition were calculated in GraphPad Prism by plotting the mean phosphate signal  versus log10 SH3 concentration using nonlinear regression using the model: One-Site - Fit LogIC50 and constraining the top and bottom values to the maximum (zero SH3 inhibition) and minimum (zero DLC1). Hydrolysis of GTP by Ras was performed similarly, using 5 μM purified H-Ras and 0.075 μM p120RasGAP GAP domain, in the presence of p120RasGAP SH3 domains at 0, 1, and 10 μM. For experiments using p120RasGAP SH2-SH3-SH2 domain, the hydrolysis reactions were performed using 200 μM GTP for 30 min, and color development for 20 min using the BIOMOL green malachite reagent (Enzo Life Sciences).Hydrolysis of GTP by RhoA was monitored in an endpoint assay using the malachite green reagent  or BIOMOL Green (Enzo Life Sciences)). All assays were performed with purified RhoA at 5 μM in assay buffer with a final composition of 20 mM Tris-HCl pH 8.0, 250 mM NaCl, 1 mM MgClFurther information on research design is available in the Supplementary InformationReporting Summary


Gluten-free rice flour noodles with a flavor and texture profile preferred by the Japanese people were developed. The rice noodles contained potato starch (PS) as a binder. “Koshihikari” was selected from several candidate varieties based on its pasting properties. Since the Japanese people prefer the chewy texture of wheat flour “Udon” noodles, first, the stress−strain characteristics of “Udon” noodles in Japan were quantified, using a mechanical test. Next, different formulations of rice noodles were prepared by changing the amount of PS blended into the noodles. The mechanical tests on wheat and rice noodles show that rice noodles made from 85% rice flour and 15% PS have a texture similar to that of “Udon” noodles. Brown rice noodles containing roasted brown rice flour were also developed. Since brown rice flour hinders the binding of the dough, it was necessary to increase the amount of PS to increase the binding of roasted brown rice flour. Finally, noodles with 70% white rice flour, 10% brown rice flour, and 20% PS were produced. The gas chromatography−mass spectrometry analysis of the volatile compounds contained in white rice noodles and brown rice noodles identified the volatile compounds characteristic each of type. Rice is a staple food of the Japanese population. However, according to data released by the Japanese government, rice consumption per capita has fallen to less than half in the last 50 years . This maIncreasing the intake of wheat flour foods by Japanese people may increase the number of people with wheat allergies and celiac disease (CD) due to gluten, which is a hydrate of the protein contained in wheat flour . Celiac Many studies have been performed on the development of gluten-free noodles. Martil et al. extracted starch from parboiled rice flour and produced gluten-free pasta via an extrusion cooking process . They reToyokawa et al. stated that Japanese people prefer a unique texture; that is, when noodles are chewed, they want to feel a softness but also an elastic resistance . In our In this study, we first investigated the mechanical properties of wheat udon noodles using several kinds of commercial dry udon noodles, and we identified the parameters that are indicators of a chewy texture, which is a texture preferred by the Japanese people. Next, we selected the rice flour variety to be used for making rice noodles via pasting properties, using a rapid visco analyzer (RVA). Noodles made with 100% rice flour without pressure extrusion absorb less water, preventing the noodles from holding their shape. In this study, we used potato starch (PS) as the binder, as it has a higher water-holding capacity than other starches ,18,19.One of the selected rice flour varieties was used to make noodles with different ratios of PS. We then compared the mechanical characteristics of the cooked rice flour noodle with those of cooked udon noodles to identify the components of rice flour noodles that most suit the texture preferences of the Japanese people.We also developed brown noodles containing brown rice flour to increase the variety of noodles available on the market. Although brown rice bran is healthy, it has a unique odor, and it is difficult to use as food. Therefore, roasted brown flour was selected. Brown noodles contain rice flour, roasted brown rice flour, and PS. Another factor relating to the quality of noodles is aroma. Much research on the aroma properties of wheat flour noodles has been conducted. Narisawa et al. analyzed the volatile compounds derived from noodles made from several wheat varieties using headspace solid-phase extraction gas chromatography–mass spectrometry (HS–SPE–GC–MS), discussed the differences in aroma characteristics, and identified compounds characteristic of udon noodles ,21. RiceIn this study, with reference to the above works, we analyzed the volatile compounds of the white and brown rice noodles, using HS–SPE–GC–MS, and we identified the volatile compounds that were characteristic of each noodle.Commercial dry noodle products  were purchased at a supermarket. The dry noodles were cooked for the standard time described on the package.Based on the ease of procurement of rice, six varieties from the Fukushima Prefecture and one variety from Niigata Prefecture were used as candidates . In the For the development of brown rice noodles, brown rice was ground and roasted  by consignment manufacturing to obtain brown rice flour.The PS was purchased from a starch maker . It was blended into rice flour (white or brown) in various ratios. Mixed flour was used for noodle making.The rice flour noodles  used for the test were manufactured on consignment using a commercial scale process . Rice flour (white or brown flour) and PS were mixed in various ratios. Tap water was added at 44% by mass to the flour. After mixing, the dough was passed into a roller 5 times until a smooth and elastic sheet was formed; this was aged in a refrigerator overnight, cut, steamed, cooled, and vacuum-packed.Water (1 L) was boiled, and approximately 20 g of noodles was cooked for different lengths of time. The texture of noodles is affected not only by its composition but also by the cooking duration. Boiling the noodles for a short duration leads to a firm texture, whereas cooking for a longer time results in a softer texture. To ensure that the water content of the cooked noodle samples, which were boiled for different periods, was the same, the contents were measured to determine the relationship between the boiling time and water content. The water content shows an approximately linear relationship with the boiling time in a realistic boiling time range. The boiling time at which the water content of the rice noodles was 70% (wet basis) was determined from the obtained relationship. The water content was determined from the weight of the noodles before drying and that after drying at 130 °C for 2 h, having allowed them to cool in a desiccator for 30 min.A blade  simulating a human tooth was attached to a material test device  and perpendicularly lowered onto a noodle string placed on a horizontal base fixed to the device used to perform the shear test. The lowering rate of the blade was 10 mm/min. The shear test was carried out within 30 min of cooking. Each sample was analyzed six to eight times. Generally, udon noodles have a rectangular cross-section, and in the mechanical tests, the blade penetrated vertically toward the longer side. The length of this longer side was called the “width” of the noodles. The “width” of the cooked noodles was measured using a vernier caliper set on the base prior to each test. The product of the thickness of the blade and the width of the cooked noodles was designated as the apparent bottom area, and the stress value was obtained by dividing the generated force by the apparent bottom area. The distance from the contact point of the blade with the noodles to the base was defined as unity, and the change in this distance was referred to as the strain. The texture of the noodles was evaluated based on the stress–strain curve.The candidate rice varieties were examined based on their pasting properties, which were quantified using the rapid visco analyzer .A sample of rice noodles (3.50 g) with 14% water content was weighed and suspended in 25 mL of water, and then, pasting properties were analyzed using the RVA. The measurement cell was held at 50 °C for 1 min, heated from 50 °C to 93 °C for 4 min, held at 93 °C for 7 min, and then cooled to 50 °C for 4 min, before finally being held at 50 °C for 3 min, based on the procedure of Nakamura et al. . Each saA quadrupole GC−MS  was used for the analysis of the volatile compounds present in the noodles. A DB-WAX capillary column  was used for gas chromatography, and high-purity He (purity 99.9995%) was used as the carrier gas (flow rate: 1.5 mL/min).An uncooked noodle sample (3.0 g) was placed in a 20 mL vial, and the headspace gas in the vial, which was thermally equilibrated at 80 °C for 20 min, was absorbed onto a solid phase  by pressurizing the vial at 140 kPa in an autosampler . After absorption, the solid phase was dried by flowing He for 1.5 min and then heated at 200 °C for 3 min to desorb and inject gas into the GC column. The GC oven temperature was maintained at 40 °C for 10 min, increased to 220 °C at a rate of 5 °C /min, and maintained at 220 °C for 10 min.An n-alkane reagent  was used to determine the Kovats’ retention index (RI) , and 1 mThe obtained chromatogram was deconvolved using the automated mass spectral deconvolution and identification system , Gaithersburg, MD, USA), and candidate compounds were identified using MS-Search  . For eacp = 0.01). A principal component analysis (PCA) of the volatile compound data was performed using the 64-bit statistical analysis tool and Excel 2010  [Tukey’s HSD test was applied for the comparison of yield stresses between cooked rice noodles using R version 3.1.3  . Statist, Japan) . The PCA, Japan) . The “prThe primary aim of the study was to develop rice noodles that suit the taste preferences of the Japanese people, while trying to simultaneously achieve a texture similar to that of udon noodles. Therefore, in order to identify the numerical index of a chewy texture, we examined the texture of udon noodles.The representative stress–strain characteristics of five kinds of cooked commercial udon noodles are shown in A representative RVA measurement result (Koshihikari flour) is shown in The relationship between the breakdown and consistency values of rice flour samples is shown in The “Sasashigure”, “Hitomebore”, and “Koshihikari” varieties from Fukushima Prefecture were selected as the candidate varieties (circled red dashed line) because of their relatively large breakdown values and low consistency values. We decided to use “Koshihikari”, which has the lowest consistency value among the three candidate varieties; that is, it is resistant to retrogradation, according to various analyses and tests. 4 mPa s, SD1448) and a medium consistency value . Here, SD means standard deviation. These data were not plotted in In addition, brown rice flour showed small breakdown  and consistency  values, and PS showed an excessive breakdown value -2-nonenal. In particular, alcohols and aldehydes have been shown to contribute to the aroma of rice . On the In addition, the signals of the volatile compounds in the cooked rice noodles were weak, and it was hard to sufficiently compare the white rice noodles with the brown rice noodles. Therefore, in this study, we analyzed the aroma characteristics of the noodles before cooking. The analysis of the aroma of the noodles after cooking is an issue to be addressed in the future, with the examination of analysis conditions. Furthermore, the scientific sensory evaluation of aroma could not be carried out due to the limitations of human resources. Sensory tests will be performed in further studies.In this study, we aimed to develop gluten-free rice noodles with a texture and flavor profile preferred by the Japanese people. The most appropriate raw rice flour for the noodles was selected based on its pasting properties. PS was used as the binder, and the blending ratio was set to 15% based on the stress–strain characteristics of the cooked noodles and upon comparison with those of udon noodles. In addition to the white rice noodles, as a product variation, brown rice noodles containing roasted brown rice flour were also developed. Both white and brown rice noodles were evaluated for their aroma, and the two types exhibited different aroma characteristics. We are currently offering prototypes of our developed product to the market, and they have been well-received. We believe that these products will enhance the gluten-free food industry.Issues to be addressed in the future include scientific sensory evaluations of the texture and aroma characteristics, GC–MS analyses of the cooked rice noodles, and the development of brown rice noodles with better texture and flavor. These challenges will contribute to further improving the quality of gluten-free rice flour noodles.


ANK1 gene that correlated with the hereditary spherocytosis phenotype. This mutation, resulting in L1340P substitution (HGMD CM149731), likely leads to the changes in the conformation of the ankyrin ZZUD domain important for ankyrin binding to spectrin. Here, we report the molecular and physiological effects of this mutation. First, we assessed the binding activity of human β-spectrin to the mutated ZZUDL1340P domain of ankyrin using two different experimental approaches—the study of association and dissociation responses of the spectrin–ankyrin binding domain and a sedimentation assay. In addition, we documented the changes in morphology caused by the overexpressed ankyrin ZZUD domain in human cell models. Our results prove the key role of the L1340 aa residue for the correct alignment of the ZZUD domain of ankyrin, which results in binding the latter with spectrin within the erythrocyte membrane. Replacing L1340 with a proline residue disrupts the spectrin-binding activity of ankyrin.Previously, we reported a new missense mutation in the  The function of the erythrocyte membrane skeleton has been studied for years. Many interactions between membrane skeleton proteins as well as membrane skeleton proteins and integral membrane proteins have been discovered through the identification of naturally occurring mutants that are mostly associated with hemolytic anemias ,2,3. TheHereditary spherocytosis (HS), reported worldwide and characterized by the presence of spherical-shaped erythrocytes (spherocytes) on the peripheral blood smear, is the most common inherited anemia (1 in 2000 births) in individuals of Caucasian ancestry ,12. Thishttp://www.hgmd.cf.ac.uk, accessed on 19 October 2022). To date, 57 different variants in the SPTB gene associated with the HS phenotype, including ten deletions, six insertions, twelve missense, fifteen nonsense, and eleven splicing mutations, were reported . Interestingly, it is relatively easy to characterize the possible mutations in human HS of the gene SPTB, which are located within the ankyrin-binding domain: repeat 14 of β-spectrin—R 1756X [ANK1 gene mutations, including 12 missense and 27 nonsense mutations, have been reported , whereas 23 mutations were identified in the ZZUD tandem of ankyrin-R, including eight variants located in subdomain ZU5A, seven in ZU5B, three in UPA , and five in the death domain.Hereditary spherocytosis is, therefore, caused by mutations in one of the genes encoding RBC membrane proteins ,20. Over—R 1756X  and repe—R 1756X . This do—R 1756X ,26,27,28—R 1756X ,30. In hANK1 gene correlated with the HS phenotype in a three-generation Polish family with autosomal-dominant hereditary spherocytosis [https://www.uniprot.org/uniprotkb/P16157/entry, accessed on 6 October 2022) [D value or abrogated binding completely [ANK1 gene encoding the erythrocyte membrane protein ankyrin-R that leads to substitution with L1340P. Our data provide new insight into the regulatory mechanisms of the membrane skeletal proteins’ interaction pathways.In our previous study, we reported a new missense mutation in the ocytosis . This muocytosis  and is ler 2022) . MoreoveEscherichia coli XL1-Blue strain were from Stratagene (USA), and the plasmid pRSETC, E. coli DH5α strain, and E. coli BL21(DE3)pLysE strain were from Invitrogen (USA).The plasmid pEGFP-C1 and the cloning host ANK1 gene (NM_020476.3:2815–4560 bp) containing ankyrin ZZUD was synthesized at GenScript , employing E. coli codon optimization within the pUC57 plasmid and subsequently cloned into the pGEX-6P-1 high-expression vector using Bam-HI and Xho-I restriction enzymes and a standard ligation protocol. The GST-ZZUD construct was amplified in XL1-Blue E. coli, and positively screened plasmids were sequenced and transferred into the expression BL21(DE3)pLysS E. coli strain using a standard protocol. AnkBD of the spectrin 14–15 repeat was previously cloned into the pRSETC expression vector proceeded by a sequence encoding Green Fluorescent Protein obtained from the pEGFP-C1 [The ankyrin ZZUD supramodule was previously described as important for spectrin binding by ankyrin . The 174E. coli strain XL1-Blue. The resulting plasmids bearing the mutated gene were sequenced to verify the mutations. The plasmid GST-ZZUDL1340P was then transferred into BL21(DE3)pLysS cells for overexpression of the proteins.Site-directed mutagenesis was performed with the QuickChange Site-Directed Mutagenesis Kit (Stratagene). The PCR products were stored in the The overexpressed proteins GST-ZZUD, GST-ZZUDL1340P, and His6)GFP–AnkBD were purified using Glutathione Sepharose 4B  or Talon (Clontech) resins accordingly, as described previously GFP–AnkBD. The pur2 dmol−1) values, as previous described [CD measurements were performed on a JASCO J-1500 CD spectrometer using a thermostat-controlled cell with a 0.2 cm path length from 20 to 70 °C in 5 °C increments within the range of 205–260 nm. The ellipticity values from the CD spectra were converted into molar residue ellipticity  were prewetted in 0.2 mL TRIS buffer  for at least 10 min. Subsequently, the equilibration step with TRIS buffer was performed for 60 s, then Ni2+-NTA biosensor tips were non-covalently loaded with His(6)GFP–AnkBD of spectrin for 600 s followed by an additional equilibration step (250 s) in TRIS buffer. The association step of His(6)GFP–AnkBD with GST-ZZUD and GST-ZZUDL1340P batches in a concentration of ZZUD (range 0–2.5 µM) was carried out for 300 s. Dissociation was the last step, and it lasted for 300 s. The response was measured as a shift in the interference pattern (in nm) and is proportional to the number of molecules bound to the surface of the biosensor. To analyze the association and dissociation responses, the curves were baseline corrected with the Octet Software (Version HT 11.1.0.25) and compared in terms of their amplitude for GST-ZZUD and GST-ZZUDL1340P.The Octet K2 2-channel System  was used for the binding studies. The experiments were performed in black 96-well plates . The total volume of each sample or buffer was 0.2 mL per well, and the shaking settings for every step were 1000 rpm. The test was performed at 30 °C. Before each assay, Nig for 5 min, resuspended in elution buffer with 75 mM reduced GSH, and centrifuged again. The amount of bound His(6)GFP–AnkBD in the supernatant was determined using a Varian Cary Eclipse Spectrofluorimeter. The control experiments were performed using the Glutathione Sepharose 4B resin equilibrated in the test buffer and incubated with corresponding amounts of His(6)GFP–AnkBD. The bound values were obtained by subtracting the control values from those for His(6)GFP–AnkBD bound to the resin saturated with GST-ZZUD or GST-ZZUDL1340P. The binding parameters were identified using one site-specific binding fitting .The GST-tagged recombinant spectrin-binding domain of ankyrin (GST-ZZUD and GST-ZZUDL1340P) was adsorbed on a Glutathione Sepharose 4B resin via a GST tag. Equal volume aliquots of resin saturated with a GST-tagged protein were incubated with increasing concentrations (range 0–2 µM) of His(6)GFP–AnkBD of erythrocyte spectrin in the binding buffer  for 30 min at room temperature with gentle agitation. The resin was then spun down at 1000× 2, pH 7.4), centrifuged , and washed in the same buffer until pale pink. The ghosts were resuspended in resealing buffer  and incubated for 60 min at 37 °C in the presence or absence of recombinant ZZUD or its mutants . After resealing, the suspension was centrifuged , and the pellets and supernatants were collected for further analysis. The resealed ghosts were stained with lipophilic dye, Vybrant DiD (Molecular Probes), according to the manufacturer’s instructions, and observed on a fluorescent microscope (Zeiss AXIO), as described previously [Fresh blood samples were collected from healthy human volunteers  using anticoagulant . RBCs were washed three times with 10 mM Tris-HCl buffer (pH 7.4) containing 120 mM KCl. The intact cells were lysed in ice-cold lysis buffer  or mRFP-C1 (Addgene 54764) transfection vector coding for Green/Red Fluorescent Protein as a tag protein. The primers used for the PCR cloning are shown in The HEL cell line was a gift from former Professor Marcin Majka from the Jagiellonian University School of Medicine and kept in our laboratory since 2010, as were K562 cells that were a kind gift from Professor D. Duś from the Institute of Immunology, Polish Academy of Sciences in Wrocław, Poland. The cell lines were regularly checked for Mycoplasma contamination. Before transfection, the HEL or K562 cells were grown directly on cover slips for 8 h in 6-well, 35 mm plates  in RPMI 1640 and IMEM medium, respectively, with 10% FCS and antibiotics (100 I.U./mL streptomycin and 100 μg/mL penicillin), then transferred to medium without antibiotics. The cells were transfected with a Lipofectamine 2000 transfection reagent (Thermo Fisher) (μL) to DNA (μg) ratio of 3:1 mixture and cultured. Fluorescent microscopy assessed the changes in morphology resulting from the overexpression of WT or mutated ZZUD fragments after 48 and 72 h (Zeiss AXIO).https://www.uniprot.org/uniprotkb/P16157/entry, accessed on 6 October 2022): two tandem ZU5 domains , subdomain UPA (129 residues), and DD  (The binding activity of β-spectrin was assigned to the ZZUD domain (ZU5-ZU5-UPA-DD) of ankyrin. ZZUD arranged into the following tandem (NP_065209.2 ankyrin-1 isoform 1: 913-1487 esidues) . RepeatsTwo independent approaches were used to measure and compare the binding activity of the spectrin to the ZZUD domain of ankyrin or its mutated version, ZZUDL1340P.In a BLI assay, we measured the association and dissociation responses of the ZZUD domain of ankyrin or its mutant to AnkBD of spectrin immobilized to the sensors using the two-channel Octet K2 system A. We obsD = 1.039 ± 0.27 µM indicates that AnkBD of spectrin and the ZZUD domain of ankyrin complexes of high affinity are formed, and the value is similar to earlier values reported by others [D due to the very low fluorescence values obtained (Bmax approx. 5-times lower than in the case of the wild-type domain). These results showed that the presence of the L1340P mutation in the ZZUD domain significantly disrupts the ability to form a high-affinity complex. Moreover, the results obtained from the ZZUDL1340P fragment further confirm the importance of the leucine residue in the proper binding of spectrin with ankyrin, as we suggested previously [To obtain more quantitative data on the influence of the L1340P mutation on spectrin–ankyrin interactions in parallel to BLI, we employed a sedimentation assay B using Gy others ,35 or byeviously .m of the mutant was higher than that for the WT ZZUD domain (The circular dichroism (CD) analysis confirmed that the loss of binding was not caused by the instability of the mutant. The actual TD domain . Both prD domain . The recD domain . Slight The effect of encapsulation of the WT or the mutated ZZUD domain of ankyrin in the erythrocyte ghost on their morphology was investigated. For this purpose, red blood cells were prepared, loaded with the purified wild-type ZZUD domain and its L1340P mutant, and then the resealed ghosts were stained with DiD dye. It was observed that the ghosts of the erythrocytes loaded with the non-mutated ZZUD domain were characteristically deformed, while those loaded with the purified mutated ZZUDL1340P domain did not change their shape . These rTo assess the effect of overexpression of the spectrin-binding domain of ankyrin ZZUD on living cells in vitro, HEL and K562 cells were transiently transfected with vectors carrying inserts encoding protein fragments fused with GFP or RFP as described in the Materials and Methods section. The changes in morphology resulting from the overexpression of ZZUD or mutated ZZUDL1340P fragments of ankyrin were observed with fluorescent microscopy.For comparison, the constructs carrying the spectrin-binding domain or its L1340P mutant were overexpressed in the erythroblast cell line HEL. The results are presented in Similar observations were made using the myeloid cell line K562 B. The ovThese results indicate that the wild-type ZZUD domain, when transiently overexpressed in living cells, induced dramatic changes in the spectrin-based membrane skeleton, leading to substantial changes in cell morphology, while no effect was observed for the ZZUD L1340P mutant see also C.In hereditary spherocytosis (HS), the inappropriate binding of spectrin to ankyrin is highly correlated with the severity of the disease ,25. The The structural studies indicate that the second ZU5B domain is involved in the formation of an auto-regulating supramodule of ankyrin, ZZUD . Based oThe main factor in the correct binding of ankyrin and spectrin is the presence of complementary charged surfaces on these proteins that match each other . PatholoANK1 gene that correlated with the HS phenotype [D values of the ZZUD domain–AnkBD of spectrin complexes roughly corresponded to the values obtained previously for interaction between spectrin and ankyrin by different teams [In our previous study, we described a new missense mutation resulting in L1340P (HGMD CM149731) substitution in the nt teams . The binIn our next experiment, the lack of the ability of the mutated fragment of ZZUD to compete for binding with erythrocyte ghost spectrin was demonstrated. The absence of changes in the erythrocyte ghost morphology in the presence of the mutant form suggests that the mutant L1340P does not compete for spectrin with native ankyrin. Thus, the amino acid residue at the 1340 position is critical in maintaining the mechanical properties of the membrane and the shape of the erythrocytes. Moreover, in cellular models, we demonstrated changes in the morphology of the cells overexpressing the ZZUD domain of ankyrin. In the transfected erythroblast cell line, HEL, and the myeloid cell line, K562, the expression of GFP/RFP-ZZUD proteins causes large extensive granulation along the membrane surface of both cell types. Probably, the spectrin-binding fragment of ankyrin is competitive for endogenous spectrin in ankyrin binding activity. It should be noted that with time, the number of cells overexpressing the ZZUD domain dramatically decreased. This was probably caused by the enhanced mortality of these cells due to the presence of more ankyrin fragments, which destabilized the membrane. We observed a similar phenotype previously in HeLa cells overexpressing the binding domain of spectrin [In summary, our in vitro and in-cell results prove the key role of L1340 in the UPA domain for the proper function of the ZZUD domain, resulting in the ability to form a bond between spectrin and ankyrin in the erythrocyte membrane. Replacing L1340 with a proline residue disrupts the activity of ankyrin to bind spectrin, which is correlated with the HS phenotype of patients with autosomal-dominant hereditary spherocytosis. Thus, the hypothesis concerning the effect of a mutation was confirmed via experiments filling the gap between the genetic data and the physiological effect observed at the level of the whole organism.


Visual patterns can evoke marked, even beautiful motion illusions even if they arestatic; eye movements in all likelihood serve as temporal modulators. This paperconcentrates on Ouchi-type “relative” or “sliding” motion illusions. It outlines aneye-motion-evoked motion-blur hypothesis, which does not correctly predict the shiftdirection of maximal illusion. This failure led to a nearly new particularly simplestimulus: an arrangement of dashed lines that strongly evokes a relative motion illusion,the “orthogonal dotted lines sway.” The latter is well explained by motionintegration. There are many interesting variants (see below), and there seems to be no fullyaccepted explanation in the literature, although the motion integration hypothesis comesclosest  groups. These groups seem to move relative to each other, and—rule ofcommon fate—thus segregate into disparate gestalten. The term “relative motionillusion” seems appropriate.With steady fixation no illusion occurs.Motion, especially irregular motion, of the presenting paper or screen, or headmotion, evokes and/or amplifies the illusion.http://www.psy.ritsumei.ac.jp/∼akitaoka/ECVP2010poster.jpg>), (There are simple manipulations which modulate the illusion. An example is the “SpineDrift” illusion (r.jpg>), ; there rr.jpg>),  for a de2 and more, which is enough.The illusion strength increases with luminance; for strongest effect it is suggestedto view the examples at relatively high luminance; computer screens (laptop ordesktop) typically now have 100 cd/mIn his review of the Ōuchi illusion, Rapid movement of visual patterns on the retina leads to motion blur, which isperceptually suppressed .Motion blur can affect different parts of the stimulus pattern differently. This isillustrated by applying directional, or anisotropic, blur  should display themotion illusion which indeed is the case .Bitmap-images were calculated using Cappuccino  and subjIf we apply motion blur to the “Drifting Spines” illusion, we again find differentialeffects of the blur direction on the center and surround . As it hhttp://www.psy.ritsumei.ac.jp/~akitaoka/togetogedriftillusion.html><;many are quite intricate in shape. Thus, I pursued the question of which might be thesimplest local shapes that would exhibit a relative motion illusion,working from the above hypothesis  to maximizetexture contrast for pattern shift. It turned out that simple dashed lines—each “dot” beingsquare and alternatively black and white on a gray background . So I added a line width controller to  led to construction of the possibly most basic relative motionillusion , top. Asoller to  and it tThe various relative motion illusions are phenomenologically quite similar—they all lead tothe “swimming,” “sliding,” or “swaying” percept. They have in common that eye movements areinvolved (or other means that shift the pattern across the retina), which might lead tophenomenological similarity. Quite possibly, different mechanisms lead to the seemingdifferential motion percept e.g., . For insThe motion integration bias would also account for the orthogonal dotted-line sway , top. WhIn summary, motion-blur might play a role for perceptual effects since it leads to markedmodulation of texture contrast as shown here, and it might explain relative motion illusionswithout differentially oriented edges  and d.;