Recent electrochemical energy conversion devices require more advanced proton conductors for their broad applications, especially, proton exchange membrane fuel cell (PEMFC) construction. Covalent organic frameworks (COFs) are an emerging class of organic porous crystalline materials that are composed of organic linkers and connected by strong covalent bonds. The unique characteristics including well-ordered and tailorable pore channels, permanent porosity, high degree of crystallinity, excellent chemical and thermal stability, enable COFs to be the potential proton conductors in fuel cell devices. Generally, proton conduction of COFs is dependent on the amount of water (extent of humidity). So, the constructed fuel cells accompanied complex water management system which requires large radiators and airflow for their operation at around 80 °C to avoid overheating and efficiency roll-off. To overcome such limitations, heavy-duty fuel cells require robust proton exchange membranes with stable proton conduction at elevated temperatures. Thus, proton conducting COFs under anhydrous conditions are in high demand. This review summarizes the recent progress in emerging COFs that exhibit proton conduction under anhydrous conditions, which may be prospective candidates for solid electrolytes in fuel cells. Characteristics of COFs and their proton conducting mechanisms. Typically, a prototype fuel cell consists of electrolytes sandwiched between two electrodes namely anode and cathode. Fuel cell produces electrical energy by means of consumption of fuel  and oxygen, leaving water and heat as by-products. In principle, the generated electrons flow through circuit externally reaching cathode by producing electricity . On the other hand, generated protons have to reach cathode by passing through the electrolyte . Several classes of materials such as metal–organic frameworks,3 covalent organic frameworks,4 conjugated microporous polymers,5,6etc., were demonstrated as proton conducting materials. Among them, covalent organic frameworks (COF) are considered as potential candidates for their unique characteristics including but not limited to light weight elements attached through covalent bonding, high proton conduction along 1D nano channels, wide variety of customizable building blocks to tune structural and functional features.7–9 COFs are 2D or 3D crystalline materials tailored by lighter elements such as boron, carbon, nitrogen and oxygen, through covalent bonds in a periodic manner.10,11 Further, they exhibit high thermal stability, membrane processability, and excellent stability in harsh environments like strong acidic/basic conditions. Generally, COFs have high synthetic flexibility and structural diversity over organic porous materials and possess high stability over metal–organic frameworks. COFs generate unidirectional porous channels that provide high surface area and low density. COF skeleton is regulated by the linker size, and its symmetry. Further, linker symmetry determines the pore size, pore volume and their stacking pattern. They are counterparts of metal–organic frameworks (MOFs) in which transition metals attached to organic groups through coordination bond.12,13 Recent successes witnessed COF's flourishing potential for applications in diverse fields such as gas adsorption and separation,14 catalysis,15,16 sensors,17 electrochemical energy conversion and storage,18,19 and optoelectronics.20–23In the recent decades, development of renewable energy technologies aims to reduce the dependence on conventional fossil fuels for global energy demands, provide an alternative and sustainable way to meet the energy crisis. Among them, fuel cell technology grabs immense attention in the recent years owing to their unique characteristics such as high energy conversion efficiency, very low to zero hazardous carbon emission, fuel flexibility and long durability.24 However, high cost of synthesis and steady decline of ionic conduction at operation condition of fuel cells such as higher temperature (over 120 °C) and lower RH (<50% RH) restrict their viability for large scale commercial applications.25,26 Further, higher humidity decomposes the Nafion membrane over the period of time owing to variable temperature. In this scenario, COFs emerged as a potential candidate for solid-state electrolyte materials in proton conducting membrane fuel cells. Many COF materials account for high proton conductivity on par with that of commercial Nafion. In general, COFs are considered as electrical insulators.27 So, COFs are doped with proton sources like H3PO4, H2SO4, ionic liquids, etc. to ensure the availability and efficient conduction of protons in their skeletons.28–33 As a result, proton conduction in 1D pathway along the pores of COF skeleton is made possible. In addition, COFs as solid electrolytes selectively conduct protons from anode to cathode in fuel cells. Further, COFs do not allow the passage of electrons across the membrane and thus act as electrical insulators. Also, the well-defined porosity and 1D channels of COFs allow them to achieve these demanding properties. These unique characteristics attracted to deploy them in fuel cells as solid electrolytes. Generally, amorphous COFs results in poor proton conduction.34,35 Thus, it is important to retain crystallinity of COFs for higher proton conduction. So, the precise synthesis of crystalline COFs is dominated for achieving excellent proton conduction and to understand proton transport mechanism at molecular level.8,36–38 However, proton conductivity falls steeper after a short period of time owing to the leaching of acids (proton sources). To circumvent this limitation, choice of functional group present on COF and external proton source are to be chosen to tune the effective locking and hopping of proton. Nevertheless, proton conduction in such COFs is dependent on the humidity of environment which restricts their application in high temperature fuel cells especially at harsh conditions.39 In addition, they do not exhibit appreciable intrinsic proton conductivity for the practical applications at elevated temperature.40–43 Moreover, low-temperature proton exchange membrane fuel cells suffer low electrode kinetics, catalyst toxicity and complex water management. In order to overcome these limitations, high-temperature proton exchange membranes that could exhibit stable proton conduction over subzero temperatures to elevated temperatures are in high demand.44–48 Thus, COFs exhibiting excellent proton conduction over 1 × 10−1 S cm−1 under anhydrous condition are actively researched. In general, H3PO4 is widely used as proton conducting carrier in COFs owing to its non-volatile and non-toxic characteristics. H3PO4 could form dense hydrogen bonding network via P PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O⋯H–O and P–O⋯H–O interactions along 1D pores of COFs and thereby attain high proton conduction. Also, H3PO4 possesses high boiling point (∼158 °C) and thus expected to exhibit stable proton conduction under anhydrous condition at elevated temperature. Till date, proton conducting COFs under anhydrous condition is not exclusively reviewed. We attempted to compile anhydrous proton conducting COFs reported so far and comprehensively understand their proton conduction behavior by drawing structure–property relationships. In this review, we present the progressive development of COFs doped with H3PO4/triazole/imidazole/phytic acid/ionic liquids (IL) exhibiting high proton conduction under anhydrous conditions potential for fuel cell applications. Further, we discuss the comprehensive development of COFs displaying anhydrous proton conduction, current challenges and their future prospects in electrochemical energy devices  has been dominantly explored as proton conducting membranes in fuel cells. devices .49 developed proton conducting 2D aza fused π conjugated COFs as proton conducting materials  plays an important role in proton conductivity which is determined experimentally by electrochemical impedance spectroscopy (EIS) measurements. The lower Ea facilitates the higher proton conductivity. If Ea is less than 0.40 eV, the reaction mechanism is defined as Grotthuss mechanism in which migration of proton occurs along the hydrogen bonding network. On the other hand, if Ea is greater than 0.40 eV, proton conduction occurs via vehicular mechanism where the migration of proton takes place along with the proton source.Proton conduction in COFs generally takes place through intrinsic proton conducting groups present in their skeleton or external proton source doped with COFs nano pores. Mostly intrinsic proton conducting COFs exhibit lower conductivity under harsh conditions makes them undesirable for practical applications. For example, Mirica and coworkersaterials . Despite3PO4 is widely used as dopant for COFs owing to their resulting high proton mobility stemmed from extended H-bonding from three ionizable O–H bonds and low volatility (high boiling temperature ∼158 °C) which guarantees an uninterrupted operation at higher temperature. Also, we summarize COFs doped with other external proton sources exhibiting anhydrous proton conduction. Moreover, the pronounced effect of external proton source on the proton conduction of intrinsic COFs is also described.COFs doped with external proton source show considerably higher proton conduction even at high temperature (>120 °C). In principle, the integration of proton conducting carriers into the porous sites of COF leads to the activation of proton flow by the interaction between proton carriers and porous materials. In case of weak or no interaction between proton carriers and porous materials, it would result in leakage of ion carriers and thus compromise the stability of COFs as well as the fuel cell device. H50 first demonstrated azo-functionalized COFs (Tp-Azo and Tp-Stb) as proton conducting material by loading H3PO4 into the pores of COFs  doped Tp-Azo and Tp-Stb were denoted as PA@Tp-Azo and PA@Tp-Stb respectively. PA@Tp-Azo exhibited very good proton conduction over stilbene counterpart (PA@Tp-Stb) which is ascribed to protonation of azo groups and followed stabilization of counter anion, dihydrogen phosphate (H2PO4−). Besides, the interaction of H3PO4 with Tp-Azo COF was confirmed by measuring UV-vis spectra of Tp-Azo monomer treated with H3PO4 which showed red shift in absorption maximum from 380 to 496 nm owing to protonation of azo bond. PA@Tp-Azo showed the proton conductivity of 6.7 × 10−5 S cm−1 under anhydrous condition at 340 K. However, the proton conductivity was increased as the humidity increases and reaches the maximum value of 9.9 × 10−4 S cm−1 at 332 K and 98% RH. But, PA@Tp-Stb gave almost zero conductivity under anhydrous conditions while slight conductivity of 2.3 × 10−5 S cm−1 at 332 K and 98% RH. Moreover, these COF materials were chemically stable in harsh acidic conditions after impregnation of acids and retained their crystallinity.Banerjee and coworkers of COFs . The loa51 The mechanochemical synthesis was carried out by taking 1,3,5-triformylphloroglucinol (0.3 mmol), 2,2′-bipyridine-5,5′-diamine (0.45 mmol), N,N′-dimethylacetamide (DMAc) 60 μL, o-dichlorobenzene (30 μL), 6 M AcOH (15 μL) in a stainless steel jar with one 7 mm stainless steel ball and ball milled with different milling time and frequency. The optimized reaction condition is 30 Hz and grinding time of 90 minutes to obtain TpBpy-MC. On the other hand, solvothermal synthesis was carried out by charging 1,3,5-triformylphloroglucinol (0.3 mmol), 2,2′-bipyridine-5,5′-diamine (0.45 mmol), N,N′-dimethylacetamide (4.5 mL), o-dichlorobenzene (1.5 mL), 6 M AcOH (0.6 mL) in a tube. They mixed the reactants homogeneously by ultrasonication for 15 minutes followed by degassing through three successive freeze–pump–thaw cycles. Then, the tube was vacuum sealed and heated for 72 h at 120 °C. After cooling to room temperature, the reaction mixture was solvent exchanged with DMAc and washed with excess of water and acetone. Further, the material was dried under vacuum at 150 °C for 12 h to obtain the COF, TpBpy-ST. Bipyridine unit present in the COF is potentially beneficial to immobilize H3PO4 by forming hydrogen bonding and thereby migration of proton through hydrogen bond networks along the 1D nanopores. The proton conductivity of H3PO4 doped mechanically synthesized COF (H3PO4@TpBpy-MC) outperformed its solvothermally synthesized counterpart (TpBpy-ST) under similar operating conditions. Besides, the elevation of proton conductivity was observed as the temperature rose from −40 to 120 °C. Notably, the maximum proton conductivity of 2.5 × 10−3 S cm−1 and 1.98 × 10−3 S cm−1 was observed at 120 °C, under anhydrous condition for PA@TpBpy-MC and PA@TpBpy-ST respectively. Further, solid-state electrolyte conductivity of 1.4 × 10−2 S cm−1 at 50 °C was observed for PA@TpBpy-MC. Moreover, membrane electrode assembly (MEA) fabricated with PA@TpBpy-ST showed open circuit voltage (OCV) of 0.66 ± 0.02 V at 50 °C while PA@TpBpy-MC gave the OCV of 0.92 V, maximum current density of 29 mA cm−2 and maximum power density of 7 mW cm−2.Generally, COFs are synthesized by solvothermal methods. However, mechanochemical synthesis is considered to be cost-effective, and eco-friendly compared to solvothermal synthesis, but predominantly results in poor crystallinity. Banerjee's group attempted mechanochemical synthesis of COF (TpBpy-MC) containing bipyridine units that exhibit excellent proton conduction and high crystallinity .51 The m52 All the fluorinated COFs showed high water contact angle of 144° which is higher than that of polytetrafluoroethylene . Further, alkyl fluorinated COFs exhibited higher acid resistance over non-fluorinated COFs owing to elevation of hydrophobicity. The unaltered PXRD peak pattern of COF-F6 over the temperature range of 30–180 °C indicates the retention of crystalline nature which is beneficial for achieving anhydrous proton conductivity at elevated temperature  available for formation of hydrogen bonding with H3PO4via N⋯H–O interactions which eventually extended to three-dimensional multi-chain multipoint mode. Thus, TPB-DMeTP-COF stabilized its own pore structure as well as H3PO4 network along the channels. Notably, this COF is stable under harsh conditions  for 7 days and retained its crystallinity and porosity. H3PO4 was loaded into the COF to prepare H3PO4@TPB-DMeTP-COF by vacuum impregnation method and found to have phosphoric acid content of 266.6 wt% which is almost near to the maximum loading content (269.6 wt%). H3PO4@TPB-DMeTP-COF exhibited proton conductivity of 1.91 × 10−1 S cm−1 which is almost double of molten neat H3PO4 (≈1 × 10−1 S cm−1). Interestingly, proton conduction is stable for 20 h at 160 °C under anhydrous conditions. Also, proton conductivity increased as the temperature is raised from 100 to 160 °C at the interval of each 10 °C. Moreover, reduced proton conduction was observed when H3PO4 loading content is decreased. However, the magnitude of proton conductivity is higher than that of polybenzimidazoles which is attributed to dense nitrogen sites (imine-bonded) in pore walls that lead to continuous H3PO4 network at less amount of H3PO4 loading.54,55 In addition, the proton diffusion rate calculated from molecular dynamics at picosecond level was 6.28 × 10−4 cm2 s−1 at 383 K which is much higher than that for neat H3PO4 film (4.0 × 10−6 cm2 s−1) under same conditions. It is shown that high proton diffusion rate and the less reorganization energy of H3PO4 (0.12 eV) resulted in super flow of protons.Jiang and coworkers reported imine-bonded COF (TPB-DMeTP-COF) possessing dense and one dimensionally aligned nanochannels .53 It wa56 In contrast to imine linked COF, benzimidazole linked COF exhibited higher proton conductivity under anhydrous conditions. Interestingly, benzimidazole chains present in the COF activates electrostatic and hydrogen bonding interactions with H3PO4 network and thereby stabilize and lock the H3PO4 network in COF by forming H3PO4–benzimidazole complex. Single crystal X-ray diffraction studies confirmed the interaction between imidazolium cation and deprotonated H2PO4− which is beneficial for facile proton transport  in H3PO4@TPB-DMeTP-COF  which supported high rate of proton conduction. It is worth noting that H3PO4@TPB-DAPI-COF exhibited higher proton conductivity over H3PO4@TPB-DMeTP-COF at 100–130 °C. The amorphous counterpart of TPB-DAPI was also synthesized and found to exhibit lower proton conductivity which is due to higher activation energy (0.61 eV) and irregular pores that suppress the extension of hydrogen bonding H3PO4 network.Later, Jiang and coworkers reported polybenzimidazole based COF namely TPB-DAPI-COF .56 In coransport . GeneralMeTP-COF  that sho57 They demonstrated that triazine units present in COF is desirable for having strong interaction with H3PO4 and thereby suppress the reversibility of CN (imine) bonds and proton leakage. The arrangement of nitrogen atoms on pore walls facilitated the formation of dense H3PO4 networks and thus resulted in high proton conduction. COFs possessing irreversible triazine units showed strong interaction between the layers compared to their phenyl counterpart and thereby emerged as highly ordered crystalline materials  are scarcely studied. Liu et al. synthesized triazine based COF (CTF-H) and impregnated H3PO4 into it (58 The resulting COF exhibited high proton conductivity of 1.6 × 10−1 S cm−1 at elevated temperature of 150 °C (0% RH). Interestingly, this high conductivity lasts for 18 months under open air conditions which is important for practical applications. The high proton conductivity of the COF may be attributed to fast formation and breaking of hydrogen bond between nitrogen of triazine unit and OH– group of phosphoric acid (Ntriazine⋯H+⋯H2PO4− pairs). Moreover, the conductivity is higher than that of neat phosphoric acid (about 1 × 10−1 S cm−1). In contrast, H3PO4@CTF-L showed proton conductivity of 5.1 × 10−2 S cm−1 under same operating conditions which is due to the lower number of pyridinic nitrogen and the generated activated Ntriazine⋯H+⋯H2PO4− pairs . Nevertheless, the pristine CTF-H (non-doped) showed low proton conductivity in the range of 2.6 × 10−7 to 3.5 × 10−7 S cm−1 at temperature range of 30–150 °C, under anhydrous conditions which might be due to lack of free H+ carriers in the COF frameworks. The activation energy of CTF-H and CTF-L is to be 0.248 and 0.259 eV which indicates the Grotthuss-type mechanism for proton conduction. However, the absence of water at higher temperature (120–150 °C) supported the proposed proton conduction mechanism via activated Ntriazine⋯ H+⋯H2PO4− pairs.Mostly, H into it .58 The r59 Both COFs exhibited high thermal stability (>450 °C). They prepared mixed matrix membranes by embedding these COFs (40%) into polyvinylidene fluoride (PVDF). H3PO4 doped mixed membranes were analyzed for proton conductivity over 100–160 °C. At 140 °C, the maximum proton conductivity of 1.30 × 10−2 and 9.77 × 10−3 S cm−1 was observed for H3PO4@CPF-BT-1/PVDF and H3PO4@CPF-1/PVDF respectively.Jin and coworkers reported pyrimidine-based COFs (CPF-1 and CPF-BT-1) using an efficient tandem polycondensation reaction between aldehyde, acetyl and amidine under mild condition .59 Both 60 They manipulated the number of oxygen atoms (H-bond acceptors) in the porous site to modulate their proton conductivity. PyTTA-BMTP-COF possesses higher number of oxygen atom (12 O atoms) over PyTTA-DHTA-COF (4 O atoms) on its pores. PA@PyTTA-BMTP-COF exhibited high proton conductivity of 2.6 × 10−2 S cm−1 while PA@PyTTA-DHTA-COF showed 9.2 × 10−3 S cm−1 at 140 °C under anhydrous condition. The highest proton conductivity of PA@PyTTA-BMTP-COF is attributed to high density of available oxygen atoms in COF pores to interact with H3PO4 for the formation of hydrogen bonds and thus facilitated proton conduction.Zeng and coworkers developed pyrene-based 2D COFs having dense H-bond acceptors in pore channels to promote proton conduction under anhydrous conditions .60 They 61 manipulated the pore size and pore surface of COFs  decorated with choice of functional groups to realize anhydrous proton conduction over wide range of temperature from 80–160 °C  or basic groups that immobilize guest molecules for facile proton conduction (pyridine in TpPa-Py) . TpPa-SO3H exhibited the highest conductivity of 1.7 × 10−5 S cm−1 at 120 °C. However, TpPa-Py and TpPa-(SO3H-Py) showed no conductivity under the same conditions. Phytic acid exhibiting low volatility and well-matched dimensions with COF pore size was doped in to these COFs to prevent leaching of acid and to enhance proton conductivity. Phytic@TpPa-(SO3H-Py) showed better conductivity of 5 × 10−4 S cm−1 than phytic@TpPa-SO3H and phytic@TpPa-Py at 120 °C under anhydrous conditions. The higher proton conductivity of phytic@TpPa-(SO3H-Py) was ascribed to both intrinsic conduction from sulfonic acid groups and extrinsic conduction from pyridine groups that maintain immobilization of phytic acid inside COF pores.Banerjee's group reported COFs possessing intrinsic proton conducting groups (sulfonic groups in TpPa-SOTpPa-Py) .62 Also,63 They impregnated two different protic ionic liquids (PILs), dema[HSO4] and dema[H2PO4] into these COF materials by pressing them into pellets under Ar atmosphere in a glove box. After doping, these COFs retained their crystallinity. COF-C6 doped with dema[HSO4] showed about 2 order higher magnitude of proton conductivity compared to COF-C6 doped with dema[H2PO4] dopant at 1 : 1 weight ratio over the range of temperature from 40–140 °C. As expected, amount of doping pronounced a positive effect on the proton conductivity. At higher weight doping, activation energy is reduced which resulted in higher proton conductivity. COF-F6 doped with dema[H2PO4] at equal weight ratio maintained the proton conductivity for 48 h at 120 °C. Also, the highest proton conductivity of 1.33 × 10−2 S cm−1 was recorded at the weight ratio of 1 : 1.5 for COF-F6-[dema-HSO4].Zhang and coworkers developed alkyl-functionalized 2D COFs . Among t64 The tailored conducting groups (–SO3H groups) on TB-COF induced the highest inherent proton conductivity of 1.52 × 10−4 S cm−1 at 120 °C. PIL is a combination of Brønsted acid and base that can form hydrogen bond network and behaves as proton carriers. After doping with PIL 1-methyl-3-(3-sulfopropyl)imidazolium hydrogensulphate ([PSMIm][HSO4]), into TB-COF, the resulting PIL-TB-COF gave enhanced proton conductivity to 2.21 × 10−3 S cm−1 at 120 °C under anhydrous condition, due to the electrostatic interaction between PIL and TB-COF. The lower Ea for PIL-TB-COF over TB-COF indicates the role of PIL in reduction in energy barrier for H+ transfer and thereby improved proton conductivity under similar conditions. Both the intrinsic and extrinsic conduction of sulphonic acid group of COF skeleton and PIL synergistically enhanced the proton conduction. Moreover, PIL-TB-COF retained its proton conductivity almost unaltered over 72 h at 120 °C which is advantageous for practical applications.Also, Yan and coworkers demonstrated the incorporation of PILs to enhance the proton conduction of TB-COF .64 The t3PO4 and imidazole. Wang and coworkers synthesized iso-reticular thiophene-based COF materials  to explore the effect of hydrogen donor ability of imine linkages on proton conductivity  into COF exhibited reduced activation energy and thus rise in proton conductivity. The highest proton conductivity of 3.08 × 10−3 S cm−1 was observed for Im@Py-TT-COF(50 wt%) at 130 °C under anhydrous conditions which is about six times higher than that observed for 30 wt% imidazole loaded COF. DFT calculations revealed that the N–H⋯N hydrogen bonding distance between imine and imidazole is 1.932 Å for Im@Py-TT-COF, which is slightly shorter than those observed for Im@Py-BT-COF and Im@Py-BD-COF. This shorter distance and stronger hydrogen bonding was beneficial to well organization of imidazole and their hopping in the pores. Further, this hydrogen bonding was confirmed by the shift in imine stretching frequency of about 8 cm−1.Anhydrous proton exchange membranes (PEM) offer viability for operation at elevated temperatures (120–200 °C), which is advantageous for fast electrode reaction, and higher tolerance to CO poisoning over Pt catalyst. In general, the presence of azo, pyridine, azine and sulfone groups on COFs is exploited as hydrogen bonding sites for proton carriers such as Huctivity .65 They via reversible aldimine reaction , trz@TAPB-DMTP-COF0.1-BBA membrane gave the highest proton conductivity of 5.05 × 10−3 S cm−1 which is about 1.6 times higher than that of trz@COF that does not possess BBA units. The lower proton conductivity of trz@TAPB-DMTP-COF membrane is attributed to random orientation which results in inefficient proton conduction. In contrast, anisotropic orientation of 1D pores in trz@TAPB-DMTP-COF0.1-BBA leads to unidirectional proton transport with low activation energy of 0.21 eV and thus resulted in higher proton conductivity.Guo and coworkers developed 2D COFs using in plane crystallization in water/oil interfacial polymerization system reaction .66 By ad67 Phosphoric acid and 1,2,4-triazole have proton dissociation energy of 3.86 and 4.07 eV respectively. After doped into COF, weak interaction between COF skeleton and biphosphate anion (COF⋯H⋯H2PO4−) leads to decrease in proton dissociation energy which eventually results in higher proton conductivity. Particularly, the positively charged EB-COF  in the walls of COF pores compared to that of water in Nafion. Considering this in mind, Ma and coworkers developed four COFs and tuned the proton dissociation energy of proton carriers present in COF and thereby achieved higher proton conductivity.d EB-COF  had stro68 The retention of crystallinity and porosity of triazole doped TPB-DMTP-COF under harsh conditions such as about 120 °C for 7 days underscores its viability for real time applications. The proton conductivity was dependent on temperature and the maximum conduction recorded was to be 1.1 × 10−3 S cm−1 at 130 °C under anhydrous condition for trz@TPB-DMTP-COF. Further, triazole/imidazole doped TPB-DMTP-COF stored in 130 °C under sealed condition showed high thermal stability over a period of one month. The highest proton conductivity of im@TPB-DMTP-COF was to be 1.84 × 10−3 S cm−1 at 130 °C. The lower conductivity of triazole doped COF is attributed to relatively low proton concentration of triazole due to two lone paired nitrogen atoms. Further, deuterium substituted imidazole doped TPB-DMTP-COF confirmed the proton conduction occurs through transport of protons but not the migration of imidazole/triazole molecules.Jiang and coworkers developed triazole doped imine-functionalized TPB-DMTP-COF for anhydrous proton conduction at elevated temperature .68 The r69 Three different anilines containing sulfonic acid groups (–SO3H) and ethidium bromide were utilized as cationic and anionic monomers respectively, to react with triformylphloroglucinol to obtain three zwitterionic COFs with different pore widths. The resulting COFs obtained in keto form that was confirmed by a peak at 183 ppm (keto group) in cross-polarization angle spinning 13C solid state NMR spectrum. Further, the energy dispersive spectroscopy (EDS) confirms the removal of Br− ion of ethidium bromide along with hydrogen of sulfonic acid groups after washing the reaction mixture, which results in neutral COF channel. Triazole/imidazole was incorporated into XJCOF and evaluated the proton conductivity. Particularly, proton conduction is increased as the temperature rise from 120 to 160 °C. The highest proton conductivity of 1.29 × 10−2 S cm−1 at 160 °C observed for trz@XJCOF-2. Further, im@XJCOFs showed higher proton conductivity over their trz@XJCOF counterparts. The maximum proton conductivities of 4.38 × 10−2, 3.33 × 10−2, and 2.43 × 10−2 S cm−1 were observed for im@XJCOF-1, im@XJCOF-2 and im@XJCOF-3 respectively, at 140 °C under anhydrous condition. Moreover, the activation energy of im@XJCOFs was to be about 0.24 eV which is comparable to that of commercial Nafion (0.22 eV). In addition, both anionic and cationic groups present in the zwitterionic COFs could form ion pairs with triazole/imidazole dopant in the nano channels which act as proton donor and acceptor, and thus enhanced the proton diffusion rate.Ma and coworkers developed zwitterionic COFs (XJCOF) possessing both anionic and cationic groups in equimolar ratios .69 Three29,43,51,70–72 The suitable engineering methods need to be developed to validate the potentiality of these COFs in MEAs. (ii) The predominantly employed hydrothermal synthetic route for COFs involving large amount of hazardous organic solvents must be replaced with environmentally benign green synthesis methods. (iii) The stability of COFs may be challenged by external proton sources doped into COFs owing to its leaking tendency over a period of time. Thus, intrinsic proton conducting COFs exhibiting high proton conduction under anhydrous conditions are necessarily to be developed by molecular engineering of tailorable building blocks. The long term of stability of COFs exhibiting steady proton conductivity over a period of time is still need to be extensively investigated before implementing them in fuel cell devices. In future, alternative synthetic approaches  may dominate towards scalable synthesis of COFs from gram scale to kilogram scale for the industrial fabrication of real time devices. (iv) In fact, fuel crossover is an unavoidable phenomenon in fuel cells particularly at the initial stage of operation which pose a serious threat to the operation and stability of proton exchange membranes. Further, acid-leaking also deteriorates the PEM and potentially degrade the fuel cell system. So, the retention of acid within the pores of COFs needs to be achieved to overcome the loss of proton carriers in PEM and to retain the operational stability of fuel cells at high temperature.73–77 It is apparent that COFs are potential candidates exhibiting many valuable qualities, and if the above mentioned constrains addressed, COFs exhibiting proton conduction under anhydrous conditions would be an unavoidable platform for proton exchange membrane in electrochemical energy harvesting systems.Proton conducting covalent organic frameworks (COFs) are promising candidates for the solid-state electrolytes in fuel cells. Compared to other proton conducting materials suffer from low conductivity and poor stability, COFs have uniform 1D channels with permanent porosity for facile transportation of proton carriers and rigid framework-enabled stability. Interestingly, COFs exhibit high robustness towards harsh environments such as strong acid, strong base, and external humidity which guarantees their long-term stability in electrochemical energy devices. In this review, we have discussed the COFs constructed with different building blocks displaying intrinsic, extrinsic and combined proton conduction under anhydrous conditions. Particularly, we elaborated the role of external proton sources and their interaction with building blocks on the generation and active migration of protons along the pore channels. The choice of proton sources and their effective binding to keep them intact in nanopores of COFs to ensure immobilization of proton sources and thereby achieved the flow of proton. In addition, we have tabulated the recently developed COFs exhibiting anhydrous proton conduction in detail . The enhThe authors declare no conflict of interest.


Our laboratory has identified and developed a unique human-engineered domain (HED) structure that was obtained from the human Alpha-2-macroglobulin receptor-associated protein based on the three-dimensional structure of the Z-domain derived from Staphylococcal protein A. This HED retains µM binding activity to the human IgG1CH2-CH3 elbow region. We determined the crystal structure of HED in association with IgG1’s Fc. This demonstrated that HED preserves the same three-bundle helix structure and Fc-interacting residues as the Z domain. HED was fused to the single chain variable fragment (scFv) of mAb 4D5 to produce an antibody-like protein capable of interacting with the p185Her2/neu ectodomain and the Fc of IgG. When further fused with murine IFN-γ (mIFN-γ) at the carboxy terminus, the novel species exhibited antitumor efficacy in vivo in a mouse model of human breast cancer. The HED is a novel platform for the therapeutic utilization of engineered proteins to alleviate human disease. Staphylococcal protein A (SpA) is an important virulence factor found on the surface of Staphylococcus aureus. The protein has five tandem Z-domains (E-D-A-B-C) at the N-terminus and a cell wall anchoring domain at the C-terminus. These domains are responsible for interacting with known proteins, such as tumor necrosis factor receptor 1 (TNFR1) . The conGraille et al. investigated a ternary complex containing Fc, Z-domain, and Fab molecules. The model indicated that a single Z domain may simultaneously bind Fab and Fc . There hSpA Z-domains have been fused with proteins to boost their immunogenicity in vivo. When antigen-presenting cells (APCs) have surface Igs, ZZ-antigen conjugates exhibit increased presentation to an Ag-specific T-cell hybridoma  The bindWe solved the crystal structure of HED in complex with the Fc fragment of human IgG1(hIgG1Fc). HED was subsequently fused with 4D5scFv to generate an antibody-like species that could bind HER2-positive tumor cells through its single chain Fv portion and Ig antibodies with µM affinity via its humanized Z-domain. Additionally, the humanized antibody-like protein was fused with mIFN-γ to generate a new class of proteins known as HED-bodies. The HED-body displays potent ADCC activity and tumor growth suppression in vivo.As a fundamental structural component of Staphylococcus Protein A, the Z domain possesses a three-helix bundle scaffold, as characterized by NMR structures of the B-domain and its mutant forms  ,15. The A total of 430 structures with a Z-score greater than 2.0 were identified. As anticipated, the majority were Z-domains derived from bacteria or Z-domain-like affibodies. As our objective was to reduce immunogenicity when using therapeutic proteins, we considered the human origin of the proteins as a limiting factor in our structure search. We deemed it critical that the discovered protein has solvent-exposed surfaces to promote independent folding and prevent aggregation under physiological settings, among other structural characteristics. In addition, an optimal structure would possess “no” cysteine residues and display minimum gaps of primary sequence to facilitate its expression and maintain its essential structure.With these imposed search criteria, the structure model of the human alpha-2-macroglobulin receptor-associated protein  was identified and superimposed onto the domain D structure . The Z-shttps://swissmodel.expasy.org (accessed on 5 February 2023)). We then aligned the model to the SpA B-domain from the complex structure of the B-domain with the Fc fragment of human IgG1  and HED with a TEV protease site. We were able to co-express the GST-HED fusion protein with hIgG1Fc in bacteria BL21 (DE3). When we purified the GST-HED fusion protein using Glutathione Sepharose 4B, hIgG1Fc was also co-purified . CleavagThe structural details of the interaction between HED and hIgG1Fc were resolved at the atomic level. The crystallization of the purified complex allowed for the solution and refinement of its structure to 2.7 angstroms. The HED domain folds into a three helical bundle in the complex crystal. The first and the second helixes are involved in the interaction with hIgGFc, while the third helix is tightly tethered to the hydrophobic core and resides on the opposite side of the Fc fragment. The structure involved in Fc-binding is conserved in the HED and the Z-domain from 1FC2 and 4WWI, for which the mean rmsds are 0.479, 0.420, and 0.487 angstroms, respectively. Similar to the complex structures of the Fc fragment with the SpA B-domain or the SpA C-domain ,22, the The crystal structures of hIgG1Fc in complex with the HED and with the B-domain indicate that the Fc fragment interacts with the HED and B-domains in a similar manner A. As dedS33 and Q37, which are distinct from residues of 2FCWF1, reside in the inserted loop between helix2 and helix3 of HED . The cryIn addition to the Fc binding activity we created for the HED, we designed the new protein to retain specific three-dimensional structure features of the original human protein to reduce immunogenicity. For this reason, we only mutated the residues that participate in the interaction with the Fc fragment and residues that might introduce spatial effects. The crystal structure of HED could be perfectly aligned to the modeling region of hA2MRAP, with the mean rmsd of the superimposition at about 0.866 angstroms. The solvent-accessible surface of HED, except for the surface of helix1 and helix2, possess similar structural features as that of hA2MRAP D,E. The In order to utilize host antibodies to recruit host cells that would kill specific target cells through the CDC and ADCC pathways, the HED must bind to the Fc and avoid interrupting binding of Fc to Fc receptors and C1q. We determined that the interaction of the HED with IgG Fc did not affect the binding of the Fc to the Fc receptor. This event is an essential step to induce ADCC, a biological activity we wished to maintain. The complex structures of human IgG1Fc with human Fc receptor revealed that Fc fragments, after interacting with the HED domain, have a normal binding pattern to Fc receptors ,24. An iComplement activation for the CDC pathway necessitates the critical step of C1q binding to the Fc domain of Ig molecules, namely, IgG or IgM complexed with Ags. The complex structure of human Fc fragments with complementary C1q molecules was used to generate a complex model of HED, Fc, and C1q . The modWe fused the scFv of 4D5 to the Z domain to generate 4D5scFv-ZZ . In ordeWe prepared a plasmid to express the new 4D5scFv-HED by inserting the 4D5scFv and the HED into the vector pSectagA and expressed it in HEK-293T cells. The protein was purified by affinity chromatography and size exclusion chromatography. A human IgG1 Fc was also expressed and purified from HEK-293T cells. According to the filtration patterns on Superdex 200, the molecular weight of the Fc was determined to be 45 kDa and the molecular weights corresponding to two peaks of 4D5scFv-HED were 74 kDa and 46 kDa A. Based The 4D5scFv-HED and IgG1Fc were mixed at a molar ratio of 1:1 and incubated at 4 °C for two hours. The mixture was loaded onto a Superdex 200 column and only one elution peak was detected, with the calculated molecular weight of 140 kDa. Subsequently, the collected fractions under the peak of the mixture of IgG1Fc and 4D5scFv-HED were examined using 12% SDS-PAGE A. The reBiophysical binding of 4D5scFv-HED to Fc domains was characterized. We performed SPR studies using chips with several IgG species immobilized on them. As shown in To confirm that the 4D5scFv fragment in the fusion protein can still bind to p185Her2/neu without being influenced by the HED binding to Fc, we performed FACS studies. T6-17 cells that express p185Her2/neu were incubated with different concentrations of 4D5scFv-HED, washed with buffer, and then incubated with FITC-labeled immunoglobulin to examine binding. The 4D5scFv-HED demonstrated a clear concentration-dependent binding activity C. The inIFN-γ is a cytokine that stimulates multiple elements of the immune system. Our recently reported in vivo research showed that IFN-γ may significantly slow tumor growth when given concurrently with or after monoclonal antibody administration . RecentlWe determined whether the new scFv HED body species can lead to effector functions that are triggered by antibodies. Both ADCC and CDC activities were assessed using in vitro experiments. LDH release assay was used to measure the ADCC of 4D5scFv-HED-mIFN-γ against BT474 breast cancer cells . Human NStudies were undertaken to examine if the HED domain influences anti-tumor activity. A linker of (GGGGS)3 was incorporated between the HED domain and functional fusion fragments to increase the pharmacological stability of these engineered proteins. We then tested the efficacy of engineered antibodies in vitro by soft agar assay at different doses using the T6-17 mouse cell line that over-expresses human p185Her2/neu . We obseIn the past couple of years, the number of therapeutic protein products that can be used in clinical settings, such as monoclonal antibodies, cytokines, hormones, and engineered protein scaffolds, has grown substantially ,32. HighThe Z-domain was derived from the B-domain of Staphylococcus aureus Protein A. It has been observed that during treatment with biologics, anti-therapeutic protein anti-bodies or anti-idiotypic antibodies can form, which can reduce the clinical effect of the drugs . In ordeWe crystallized the HED-hIgG1Fc complex and solved its structure to determine how the two molecules interact at the atomic level. The structure of the complex showed that HED folded in a way similar to the Z-domain and interacted with the Fc fragment in the same way as the Z-domains. Except for the Phe1 residue at the N-terminal, the HED domain maintained a conserved interface with Fc, displaying an unchanged frame for main chain atoms as well as an unaltered orientation for side chains. This structural feature is critical for interaction with the Fc fragment. Consistently, residues of the Fc region involved in the interaction with the HED or Z-domain maintained the main chain structure and underwent little change for the side chain orientation. Moreover, other HED surfaces not participating in the interaction with Fc also maintained the homologous structure of the hA2MRAP fragments. This was shown in the three-structure alignment typically represented by the residues on the surface of helix2 and helix3. Residues that underwent change resided in the linker between helix2 and helix3, an area that does not exist in the structure of the 2fcw fragment. Therefore, the HED had the same structure as the original human protein it was generated from, except for the residues that formed the interface with the Fc fragment.SpA Z proteins were found to bind to the Fab of IgM. However, the structure of HED indicated that the residues on the surface of helix2 and helix3 were different from those in the Z-domain that contribute to its interaction with the Fab fragment of human IgM. Consistently, the SPR assay showed 4D5scFv-HED and, unlike the Z-domain, could not bind to human IgM. By binding to VH3 clan membrane-bound immunoglobulins, the Z-domain could induce expansion of B cells. Therefore, the humanized HED domain should have reduced immunogenicity as it lacks VH3 affinity.Cytokines are potent immune-modulating proteins that are used in cancer treatment . IL-2 isIn conclusion, based on the three-dimensional structure of the Z-domain of SpA, we constructed a novel human-engineered domain (HED) structure that was derived from the human Alpha-2-macroglobulin receptor-associated protein. The novel HED domain with Fc binding ability could serve as a promising module in a fusion protein scaffold for developing therapeutic interventions against infectious and malignant diseases.In order to create a humanized version of the Z-domain of Staphylococcus aureus protein A (SpA), the crystal structure of the D-domain of SpA in complex with the Fab fragment of human IgM antibody (PDB code: 1DEE) was used as a model to search for a similar three-dimensional structure in the Protein Data Bank on the DALI webserver . From thd-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM, and the cultures were incubated for an additional 20 h. The cells were harvested by centrifugation and resuspended in 80 mL lysis buffer containing 20 mM phosphate buffer pH 8.0, 500 mM sodium chloride, 1 mM PMSF, 1 mM DTT, and a protease inhibitor cocktail. The cells were lysed by sonication and the supernatant was separated from the pellet by centrifugation at 20,000× g for 30 min at 4 °C. Then, the supernatant solution was incubated with 2 mL Glutathione-Sepharose beads overnight at 4 °C in lysis buffer and the beads were washed 3 times using 10 mL lysis buffer and 2 times using 10 mL TEV protease digestion buffer consisting of 50 mM Tris pH 8.0, 100 mM sodium chloride, 1 mM DTT, and 0.5 mM EDTA. The mixture of beads and proteins was split into about twenty aliquots. For each aliquot, 14 mL of digestion buffer and 0.5 mL 1 mg/mL TEV was added for digestion on beads. After 8 h of incubation at room temperature, 40 μL mixture was set aside for SDS-PAGE assay and the other solution containing the complex of HED and hIgG1Fc was separated from the beads. The complex solution was pooled together and concentrated for further purification using size exclusion chromatography (Superdex 200) in digestion buffer on an Akta FPLC purifier system. The fractions containing the complex of hIgGFc and HED were collected, concentrated, and dialyzed against the buffer of 20 mM tris pH 8.0, 100 mM sodium chloride, and 1 mM DTT. Subsequently, the complex was concentrated to 18 mg/mL for further research.The cDNA for HED was synthesized and subcloned into vector PTG (which was modified from pET28a by insertion of the genes of GST and TEV Protease recognition site between Nde I and BamH I) to generate a GST fusion protein, GST-HED. The plasmid pET21a-hIgG1Fc, containing the human Fc fragment of human IgG1, was constructed from the vector pET21a. Subsequently, the two plasmids, PTG-GST-HED and pET21a-hIgG1Fc, were co-transfected to Escherichia coli Blx using 100 μg/mL ampicillin and 50 μg/mL kanamycin as selection antibiotics. A single colony was inoculated into 25 mL of Luria broth media with 100 μg/mL ampicillin and 50 μg/mL kanamycin. The starter culture was incubated and shaken at 220 rpm at 37 °C overnight, whereupon it was used to inoculate 0.75 L cultures that were allowed to grow at 37 °C to an OD of about 0.8–1.0 at 600 nm. Then, the culture was cooled down to 16 °C and isopropyl-beta-At the same time the DNAs of hIgG1Fc and novel Grababody 4D5scFv-HED with His-tag (single chain variable fragment fusion of 4D5 with humanized Z-domain) were cloned separately into pSectagA expression vectors. Then, plasmids were transfected to human embryonic kidney (HEK) 293T cells by lipofectamine 2000 using the standard transfection procedure. The target proteins were purified from the medium using Nickel-Sepharose beads after concentration. The proteins were further purified on an AKTA FPLC using Superdex 200 in PBS and stored at −80 °C. In order to test the binding of the humanized Z-domain to the human Fc fragment in solution, hIgG1Fc and 4D5scFv-HED of equal molarity were incubated together for 2 h at 4 °C and loaded on Superdex 200 of the AKTA FPLC for a binding assay. For each fraction of the peak, a 20 μL sample was loaded on a 12% SDS-PAGE and Coomassie brilliant blue staining solution was used to visualize the proteins.v/v Jeffamine M-600) at 4 °C. Diffracting crystals were formed by hanging-drop vapor diffusion after several weeks and were then directly flash-cooled in liquid nitrogen. Diffraction data were collected at 100 k on beamline A1 at MacCHESS. The diffraction data were reduced using the HKL package, and the statistics of data collection and processing are summarized in http://www.delanoscientific.com/ (accessed on 5 February 2023)).The complex of hIgG1Fc and HED was diluted to about 9 mg/mL in buffer  and mixed in a 1:1 ratio with crystallization solution  was calculated as the Koff/Kon ratio.T6-17 cells express HER2 and can be recognized by 4D5scFv. The FACS buffer  washed cells were incubated without or with the fusion protein 4D5scFv-ZZ or 4D5scFv-HED in a volume of 0.2 mL FACS buffer for about 30 min on ice. After being washed, the cells were incubated with FITC-labeled goat immunoglobulin. Then, the mean fluorescence intensity (MFI) of each sample was recorded after FACS analysis of the stained cells.2. After incubation, the supernatants were collected and subjected to LDH measurement according to the kit’s manufacturer’s instructions. LDH release (%) was calculated as ([A]sample − [A]minimum)/([A]max − [A]minimum) × 100%, where [A]max is the absorbance value of a positive control (Triton X-100) in which complete target cell lysis occurred and [A] minimum is the negative control (without effector cells).Cytotoxicity was determined with the CytoTox 96 Cytotoxicity Assay Kit  using healthy donor human NK cells obtained from the Human Immunology Core at the University of Pennsylvania as effector cells at an E:T ratio of 2.5:1. Briefly, target cells (1 × 104/well) were seeded into flat-bottom 96-well plates (Nunc) the night before the experiment. Proteins were incubated with 20 μg of human IgG1 for 30 min at 4 °C in PBS and then added into wells. NK cells were then added (2.5 × 104/well) in a total volume of 100 μL for 4 h at 37 °C with 5% COCarcinogenesis is characterized by anchorage-independent growth, which is the capacity of altered cells to expand without support from a solid surface. One of the most rigorous assays for cell malignancy is the soft agar colony formation assay, which is a well-established technique for assessing this capacity in vitro. Here, HBSS was used to make 2.5% Noble agar, which was then autoclaved before use. Later, 10% FBS in 1X DMEM  was used. The bottom layer agar was used at 0.5%. T6-17 cells were trypsinized and seeded at a density of 10,000 cells/plate and later kept at 37 °C in an incubator. Cells were later treated with media, Herceptin, 4D5scFv-HED-15-linker-mIFN-γ, and 4D5scFv-HED-15-linker-hIFN-γ at different doses every 3 days. Plates were stained with p-INT violet after three weeks and then photographed. The percentages of the colonies were later analyzed using ImageJ software 1.54a.Athymic nude mice (Jackson labs) were injected with 0.5 x106 T6-17 cells into the right flank. After a period of about 1 week, mice were randomized and injected with PBS, 4D5scFv-ZZ-mIFN-γ, and 4D5scFv-HED-mIFN-γ, respectively, every alternate day. In another set of experiments, mice were injected with 1 x106 CT26 Her2 cells. Here, the mice received PBS, Herceptin, 4D5scFv-HED-15-linker-mIFN-γ, and Enhertu as described above. Tumor volumes were measured using Vernier calipers every alternate day. Mice were sacrificed after 3 weeks. Tumor volumes were then calculated and plotted using GraphPad prism software version 6.0.Mice were housed and procedures carried out in facilities provided by the University of Pennsylvania which is AAALAC accredited. Procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pennsylvania.


All calculations presented in this study were performed using the density functional theory (DFT) and atomic gaussian type orbitals basis set. Nanotubes with chirality indices , ,  and  corresponding to diameters of 10.68 Å, 14.90 Å, 28.26 Å and 41.90 Å, respectively, are examined. Our results reveal that for nanotubes with smaller diameters, the structure obtained through rolling from a slab is not optimal and undergoes spontaneous deformation. Furthermore, this study demonstrates that the nanotube torsion deformation leads to a reduction in the band gap. This observation suggests the potential for utilizing such torsional deformations to enhance the photocatalytic activity of the nanotubes.In this study, the influence of torsional deformations on the properties of chiral WS The first WS2 nanotubes were synthesized in 1992 by Tenne [2 nanotubes. Modern synthesis methods involve a heterogeneous reaction between powdered WO3−x and a gaseous mixture of H2S and H2/N2 at temperatures ranging from 800–950 °C. Using this method, nanotubes with lengths of 1–10 µm and diameters of 20–150 nm can be obtained [Tungsten disulfide WS is a pro2-based nanotubes have been comprehensively investigated theoretically using quantum mechanics, molecular dynamics, and molecular mechanics methods. In [2 single-walled (SWNT) and multi-walled nanotubes with various chirality indices was calculated using DFT-LCAO and DFT-LACW methods. This allows obtaining the desired value of the band gap by varying the diameter of NTs [2 NT/PbSxSe1−x quantum dots heterostructures, demonstrated the possibility of ultrafast charge transfer, which is remarkable for their application as solar cells. Alternatively, one can explore the mechanical deformation of nanotubes with fixed chirality, rather than studying nanotubes with different chirality indices.Moreover, WShods. In ,5, the er of NTs . It was r of NTs ,7 indican1, n2). These indices provide information about the nanotubes’ symmetry, length of the translation vector, number of atoms in the unit cell, and diameter [n, n) and  indices are achiral, possessing inversion symmetry, while other nanotubes are chiral, exhibiting left and right screw axes [The geometry of nanotubes can be completely characterized by chirality indices  needle pushes a lever fixed at the middle of the NT, causing torsional deformation of the structure. This technique ensures accurate measurement of mechanical and electrical properties, such as the shear modulus and conductivity of the material. In addition, theoretical studies have been conducted using semi-empirical DFTB  method to examine the band structure changes of achiral WS2-based nanotubes under torsional deformations [Several studies have been conducted to experimentally investigate torsion deformations of WSubes. In ,20, a mermations . The res2 nanotubes with chirality indices n where n = 2, 3, 6, 9  were used in the calculations of the Coulomb and exchange series. These tolerances mean that during the direct lattice summations, the one-electron integrals and two-electron Coulomb integrals less than 10−8 are estimated by the multipolar expansion, and two-electron exchange integrals less than 10−16 are ignored. For more comprehensive information, please refer to the CRYSTAL17 User’s Manual [−1. Convergence criterion on the atomic displacement was set equal to 0.0001 Bohr.Ab initio calculations in this work were performed with CRYSTAL17 ,29 progrs Manual . Both th2 and a single 0001 monolayer composed of three atomic planes (S-W-S). Experimental measurements revealed a fundamental band gap of 2.4 eV for the WS2(0001) nanosheet with 1 monolayer thickness when supported by Si [2O3 [2 (1.4 eV).By incorporating roto-helical symmetry throughout the calculation, both computational and memory costs were significantly reduced. To validate the computational scheme, atomic and electronic structure calculations were performed for 2H-WSed by Si  or α-Al2 Si [2O3  substratThe results obtained by us from the quantum chemical simulations demonstrate good agreement with the experimental crystal lattice parameters and reasonable agreement with the electronic band gap, see Let us consider a brief overview of the theoretical modeling of nanotubes with hexagonal morphology using the theory of line groups. For more comprehensive information, please refer to the monographs ,36 and aStructures of tubes with a hexagonal morphology, obtained by rolling a hexagonal layer . By finding the greatest common divisor (GCD) between From a practical standpoint, it is convenient to introduce the variable In order to identify groups that describe deformed nanotube (NT) structures, it is necessary to find for each of them r values for which the torsion angle By utilizing the Formulas (3) and (4), the value of 2-based chiral nanotubes In the present paper, we consider torsion deformation of WSThe input values of NT diameters a.For all NTs in consideration, the translation vector N, E, The formation gapE) dependence on the nanotube diameter. As the energy gap for bulk is smaller than that for the slab (1.68 eV instead 2.53 eV), the band gap enlarges as the diameter of the nanotube (NT) increases. Moreover, it shifts toward the value of the band gap observed in a monolayer configuration. In the latter two lines of For each nanotube analyzed, the impact of torsional deformation was studied within the range of ngle see , and subIn It is evident from Notably, it can be observed that the physical properties of nanotubes undergo more essential changes upon torsion as the initial nanotube diameter increases. The asymmetric band gap values relative to the torsion angle 2-based nanotubes due to torsion-induced deformation. While the observed trends in band gap for  and  are comparable, it is reasonable to hypothesize that nanotubes (NTs) with larger diameters exhibit similar tendencies in electronic properties.For the nanotubes  and , the band gap decreases with torsional deformation, a trend that aligns with the findings of previous studies ,21, demoFurthermore, it is apparent that as the diameter increases, the contribution of the top of the valence band to the change in Based on the obtained data, it is evident that the nanotubes under investigation are not suitable for photocatalytic water-splitting, as they exhibit indirect bandgap semiconductor behavior.2-based chiral nanotubes using first principles analysis. We utilized line groups theory to reduce computational costs and applied density functional theory (DFT) for quantum chemical calculations.In this study, we investigated the impact of torsional deformation on the properties of WSOur findings reveal that nanotubes with smaller diameters exhibit a more significant deviation in the position of the energy minimum on the torsion curve. In this context, the smaller the diameter, the more substantial the deviation. A decrease in diameter also causes diminishing of relative diameter changes with partial translation increases, aligning with the nanotube’s stretching.Additionally, our study shows that torsion has a minor impact on nanotube diameter within the range of −2° to 2°, but leads to more significant changes under stronger torsion, especially in the case of the nanotube .2 nanotubes.We observed that torsional deformation has a limited effect on the band gap in nanotubes with small diameters but becomes more noticeable as the nanotube diameter increases, aligning with previous research . This prThe study showed that the studied nanotubes cannot be used for photocatalytic water-splitting due to the indirect band gap. However, in the case of tube , strong torsional deformation changed the band gap from indirect to direct, a phenomenon that deserves further study. Plots of band zones can be found in the


With tuning the linker of covalent organic frameworks (COFs), the “air gap” structure is oriented to shrink, leading to an increased εr of COFs and a lower Eb to facilitate exciton dissociation. Meanwhile, taking “water-/oxygen-fueled” photo-induced electron transfer reversible addition–fragmentation chain transfer (PET-RAFT) polymerization as a demonstration platform, it can be seen that COFs with a small “air gap” structure have relatively superior photocatalytic activity. This provides important implications for the evolution of efficient photocatalysts.Boosting the dissociation of excitons is essential to enhance the photocatalytic efficiency. However, the relationship between the structure of the catalyst and the exciton effect on the photocatalytic activity is still unclear as the main problem. Here, it is proposed that as a descriptive factor, an experimentally measurable dielectric constant ( The dielectric constant is acted as a descriptor to help reverse the rational design of COF-based photocatalysis with low exciton binding energy to enhance the photocatalytic activity. Compared with the high dielectric constant (εr) of inorganic semiconductor photocatalysts, the εr of organic polymer photocatalysts is small, and their strong binding may even trigger the decay of excitons.10 In particular, the interlocking iso-network structure and low-polarity periodic skeleton of COFs make the dissociation of excitons difficult.Covalent organic frameworks (COFs) comprise an attractive category of crystalline organic polymer photocatalysts with pre-programmed structures capable of fulfilling functionally targeted design requirements.εr is directly related to the polarizability of the material and depends heavily on the chemical structure of the material.16 According to the Wannier–Mott model:17Eb = EHm*/εr2 , the εr has the potential to quantitatively evaluate photocatalytic activity.18 In this regard, Wang's group inserted ionic sites into the skeleton of COFs by polarization engineering, in which highly dipolar ionic groups enhance the directional polarization of COFs, resulting in ionic COFs with high εr relative to neutral COFs and displaying more active exciton behavior.10 It is evident that the εr is a descriptive factor to quantitatively characterize its relationship with Eb along with the photocatalytic activity which is important for the development of efficient photocatalysts.9,10,19–22The 23–25 Porous materials are primarily designed to increase the porosity in the intrinsic structure of the material in order to effectively reduce the density of the material and thus decrease the number of polarizable dipoles in the interlayer medium.1,10,19,26–29 The “air gap” structure refers to where air (with a minimum εr of 1 for air) is mixed into a dense material , with the air eventually acting as the dielectric.30 Conversely, to lower the Eb, the design principle of the photocatalyst entails a target along high εr. As is known, oxygen is a radical sudden extinguisher, which greatly impedes the occurrence of controlled radical polymerizations (CRPs) and confines the industrial scalability and wide application of CRPs, and it is extremely significant to tackle this difficulty for advancing the research progress in polymer science.31Pursuant to the International Roadmap for Semiconductors (ITRS), porous materials and “air gap” structures are an effective way to develop low dielectric materials.εr and lowering the Eb. The study confirms that the εr can be applied to describe the photocatalytic activity of COFs, correlates the “air gap” structure of COFs with the dielectric properties and exciton effect, and reveals the influence law of the “air gap” magnitude on the photopolymerization reaction activity.In the present study, we take “water-/oxygen-fueled” photo-induced electron transfer reversible addition–fragmentation chain transfer (PET-RAFT) polymerization as a demonstration platform to optimize the photocatalytic activity of COFs by narrowing the “air gap” structure of COFs through ligand tailoring, enhancing the C2/m space group with cell parameters of a = 24.0733 Å, b = 24.4929 Å, c = 3.8657 Å, α = 90°, β = 91°, and γ = 90° and a = 28.7932 Å, b = 27.9868 Å, c = 3.7906 Å, α = 90°, β = 90°, and γ = 90°, respectively. Meanwhile, the experimental results of Py-TPA-COF and Py-BPDA-COF are aligned highly with the results of P-XRD simulations of AA stacking, indicating that both are arranged in AA stacking mode with a layer-to-layer spacing of 3.86 Å and 3.78 Å, respectively  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>N stretching mode of the imine, suggests the successful preparation of the target COF. 13C CP-MAS NMR spectra , meaning the successful assembly of the skeleton. Scanning electron microscopy (SEM) images  characterization incorporating theoretical structure simulations. As shown in ectively . High-reectively  enable tectively , the disa Fig. S3 with peas Fig. S4 display d Fig. S5. With thεr of Py-TPA-COF and Py-BPDA-COF was measured by using the parallel plate capacitor principle on an impedance analyzer. The εr of Py-TPA-COF decreases with increasing frequency and outperforms the εr of Py-BPDA-COF in the frequency range of 40 Hz to 10 MHz  spectra were examined. As seen from the inset of Eb.34 The fitting results showed that Py-TPA-COF (Eb = 45.0 ± 2.6 meV) has a lower Eb than Py-BPDA-COF (Eb = 53.8 ± 5.9 meV), implying a relatively better charge–hole separation of Py-TPA-COF after absorbing photon energy. The above experimental results suggest that the directional reduction of the “air gap” structure by ligand modulation can lead to an increase in the descriptor εr, which effectively reduces Eb, which is consistent with the reported correlation pattern.10The descriptor o 10 MHz . As is ke the εr . To furtN atoms of the imine linker mode. The distribution conditions of positive and negative charges are similar and uniform for both, showing no local polarization. The material density of Py-TPA-COF with a short linker is larger than that of Py-BPDA-COF, which means that more dipoles can be polarized by the alternating electric field, coupled with a smaller “air gap” structure, resulting in a larger εr  is applied to interpret the dielectric properties and exciton dissociation behavior of the different “air gap” structures. From the electrostatic potential analysis of Py-TPA-COF and Py-BPDA-COF , it is sarger εr . In the arger εr , the eleεr and thereby suppress the exciton effect enables efficient charge–hole separation and photocatalytic activity. Firstly, the light harvesting ability of Py-TPA-COF and Py-BPDA-COF was evaluated by UV-vis DRS. As seen in Eg) of Py-TPA-COF and Py-BPDA-COF were calculated to be 2.23 eV and 2.27 eV, respectively, according to the Tauc plots deduced from the Kubelka–Munk function  were determined by extrapolating the Mott Schottky curves to 1/c2 = 0.36 The Efb of Py-TPA-COF and Py-BPDA-COF was estimated to be −0.82 and −1.12 V vs. NHE, respectively  potentials of Py-TPA-COF and Py-BPDA-COF are +1.41 and +1.15 V vs. NHE, respectively (2/O2˙− (−0.33 V vs. NHE)15 suggest the feasibility of generating superoxide anions (O2˙−), and insufficiently positive potentials relative to H2O/OH˙ (2.73 V vs. NHE)38 make the direct oxidation of water by holes to form OH˙ an infeasible path. Electrochemical impedance analysis  curves  curves  record t41,42 which enabled a deep reaction. Then, the reduction capacity of photogenerated electrons of Py-TPA-COF and Py-BPDA-COF was assessed by means of EPR .45 The production of OH˙ was again demonstrated with the assistance of the UV-vis absorption spectrum of the indicator methylene blue (MB)  Fig. S8.Mw/Mn < 1.5) and have molecular weight (Mn) close to the theoretical line . The polymerization eventually goes into the RAFT cycle with increasing light time. In situ chain expansion experiments (Photo-induced electron transfer reversible addition–fragmentation chain transfer (PET-RAFT) polymerization is mild and controllable, and is a powerful means of preparing target polymers with desired molecular weight and molecular weight distribution. The fundamental solution to the problem of oxygen blocking polymerization is to consider oxygen as the feedstock for catalytic polymerization. Therefore, “water-/oxygen-fueled” PET-RAFT polymerization was examined . As showcal line . Simultaon trace  of the pon trace , which ion trace , the peaeriments  and S13†2˙− anion was quenched by a sufficient amount of BQ, the polymerization process did not occur in either case and was alleviated as the amount of BQ decreased (2˙− inevitably hinders the generation of OH˙ thereby inhibiting the polymerization reaction. Similarly, when another fuel water was neglected, the pathway of OH˙ generation was hindered (To dissect the photocatalytic reaction mechanism of Py-TPA-COF and Py-BPDA-COF, the main variables were modulated. When the Oecreased . The abshindered . Controlhindered  demonstrhindered . Not surhindered . The prehindered . The cheεr is a promising descriptor of Eb, an additional pair of TFP-COFs with different air gap structures were investigated by considering the pore size as a single factor. Analogously, TFP-1Ben with smaller air gap structures has higher εr, lower Eb and more efficient high catalytic activity relative to TFP-2Ben (Fig. S15–19εr descriptor has the potential to facilitate reverse on-demand photocatalyst design, which will provide a new strategy for the design and development of more efficient photocatalytic systems.To further evidence that the εr parameter as a descriptor to quantitatively describe its relationship with Eb and photocatalytic activity. By shortening the ligand to target the “air gap” structure of the COF, the descriptor εr is increased to lower the Eb and promote the dissociation of excitons to form free carriers to obtain higher catalytic activity. At the same time, the RAFT polymerization protocol using water and oxygen as fuel supply is explored in an open environment, which is important for the development of polymer science and the evolution of efficient photocatalysis.In summary, this work proposes an experimentally measurable All relevant data supporting this article have been included in the main text and the ESI.H. Y. proposed the research idea, preparation and characterization of the catalyst and wrote the manuscript. Z. L. explored the synthesis method of the catalyst. X. Y. proposed the test method of dielectric properties and the revision of the manuscript. S. W. proposed the idea of dielectric property testing. L. H. contributed to the revision of the manuscript and the financial sponsorship.There are no conflicts to declare.SC-014-D3SC01719B-s001


At the end of the 60‐day storage period, cricket flour treated with sodium benzoate and garlic extracts both had a significantly lowest population of yeast and molds (1.91 log cfu/g). On five point hedonic scale (1. Dislike extremely and 5. Like extremely), color (3.84 ± 0.86–2.55 ± 0.99), aroma (3.59 ± 1.09–2.40 ± 1.01), texture (4.11 ± 0.97–3.11 ± 0.97) and overall acceptability (3.77 ± 0.64–2.83 ± 1.01) sensory scores were all significantly high on day 0 and low on day 60 of storage, respectively. The study concluded that preserving crickets with garlic extracts significantly reduced the population of yeast and molds. Cricket flours were microbiologically safe and acceptable to consumers. Therefore, storage of cricket flour preserved with garlic and ginger extracts for longer periods is recommended. In addition, utilization of the preserved flour as an ingredient in different food applications is recommended to determine its suitability and sensory acceptability.Although spices have been used in food for centuries, little is known about their use to preserve insect‐based foods. This study assessed the flour produced from blanched crickets treated with extracts of either ginger, garlic or both at a ratio of 1:4 (v/w) for color, pH, microbiological profile, sensory quality, and acceptability. Sodium benzoate treated and untreated cricket flour was used as positive and negative controls, respectively. The flour was stored at ambient conditions and analyzed on 0, 30, and 60 days of storage. The pH, moisture content and color change increased during storage but remained within acceptable limits. The total microbial count, yeast and molds significantly decreased with storage duration ( At the end of the 60‐day storage period, cricket flour treated with sodium benzoate and fresh garlic extracts had the lowest microbial counts including yeast and molds. The pH, moisture content and color change remained within acceptable limits for both treated and untreated cricket flour. Consumers liked both the spice‐treated and control cricket flour samples, although acceptability declined with prolonged storage. Samples were sorted to remove bad quality, soaked in water for 30 min, washed and rinsed under running water; with care to avoid any tissue damage. Subsequently, the skin was peeled prior to crushing/grating for extraction according to previously established procedures  were obtained from insectPro farm, which is located in Limuru, Kenya. Prior to harvesting, crickets were fasted for 48 h while being given only water to cleanse their guts of the ingesta. The crickets were transported to the laboratory in aerated boxes and inactivated by freezing them at −20°C overnight. Frozen crickets were allowed to thaw at room temperature for 2 h. To remove dirt and feed residues, frozen crickets were washed three times in tap water using a cricket to water ratio of 1:3 (w/v) according to previous procedures . Prior to packaging flour from each treatment was divided in to three parts which were put in separate packages, such that a package was randomly picked for analysis on day 0, 30 or 60. Packages that were opened were not returned to storage. The samples were kept on shelf at ambient conditions; room temperature (23 ± 2°C) and relative humidity (60 ± 2%) and subjected to physicochemical, microbiological, and sensory evaluation at days 0, 30, and 60 storage periods.2.3The pH was determined using the method that was previously validated Kalra, . In brieL*, a*, b* color systems. L* represents whiteness/blackness (100 = white and 0 = black); a* represents redness/greenness ; and b* represents yellowness/blueness . Measurement was performed six times on approximately 10 g of flour in a small container. Color changes  were calculated using the samples at zero as a reference. The total color difference (ΔE*) which shows the extent of color change in parameters between the initial and final color values during storage, was calculated using the equation by  and incubated at 35 ± 0.5°C for 24 ± 2 h. Total yeasts and molds were determined on Sabouraud Dextrose Agar (SDA) plates incubated at 27°C for 48 h, as recommended  utilizing Nutrient Agar (NA) as the medium, according to previous procedures Sanders, . Prior t2.5The sensory/organoleptic properties of flour during storage were evaluated for color, aroma, texture/fineness, and overall acceptability on days 0, 30, and 60 of storage. Sensory quality was evaluated following the principles and practices previously described . Analysis of Variance (ANOVA) was performed and and means were separated using Bonferroni adjustment at 95% confidence level. Results were reported as mean ± standard deviations. PCA was applied after the Two way ANOVA to a set of data that was obtained from the color, moisture, pH, microbiological analysis, and sensory evaluation of samples from various treatments and storage intervals. The original data was transformed into a smaller set of linear combinations called principal components (PC). Two PCs were chosen, and their combination described the best treatment and storage time.33.1L*) values declined from day 0 to day 60. For both the treated and untreated samples, lightness at day 0 was significantly higher than at days 30 and 60 in storage (p ˂ .05). However, lightness values across samples at the same stage of storage did not differ significantly. Measurement for redness/greenness (a*) did not vary significantly among samples (p > .05). The highest value was observed on day 0, while the lowest value was on day 60 for all the samples. The flour from cricket preserved with garlic and garlic mixed with ginger had the highest values at day 0 . In the garlic/ginger‐treated, sodium benzoate‐treated, and untreated samples, the measurement for yellowness/blueness (b*) of the cricket flour samples varied significantly at day 0 and day 30 of storage (p ˂ .05). The total color change (ΔE) of samples from the initial storage to the final storage period differed significantly (p ˂ .05). The highest color change was noted at day 60 of storage in the untreated samples (ΔE = 11.71 ± 0.55). The color change between day 0 and 30 was minimal among samples, ranging from 2.09 ± 1.01 to 2.87 ± 1.09 in flour preserved with garlic and untreated flour.There was a significant interaction between sample treatment and storage duration for Total color change and Lightness values, as shown in Table p ˂ .05) across the various storage stages than it was within samples. The moisture level ranged from 1.21 ± 0.32% to 1.48 ± 0.11% on day 0; by day 60 of storage, it was in the range of 2.82 ± 0.08%–3.02 ± 0.46%. The pH values increased from day 0 to day 60 during storage, showing a significant difference between samples and storage time (p ˂ .05). At day 0, the cricket flour preserved with ginger‐garlic mixed extracts had the lowest pH (6.39 ± 0.00) while the cricket flour treated with sodium benzoate had the highest (6.43 ± 0.00). The sodium benzoate‐preserved samples had the lowest pH at the end of the storage period, whereas the ginger‐preserved cricket flour had the highest pH .Table 3.2E. coli and fecal coliforms were undetected, the total microbial load and yeast and mold counts decreased with storage duration (Table p > .05). Among the samples and the storage period, a significant decline in the population of yeast and molds was observed (p ˂ .05); with the untreated samples having the highest population on days 0 and 30 of storage . At the end of the 60‐day storage period, cricket flour treated with sodium benzoate and garlic extracts had the significantly lowest population of yeast and molds (1.91 log cfu/g).While on Table . The TVC3.3p ˂ 0.05). The rating for color decreased with an increase in the number of days in storage. Similarly, the aroma of the treated and untreated cricket flour decreased significantly with the length of time in storage (p ˂ .05); and the aroma was the least rated of all the parameters. Texture was rated higher than other sensory attributes in all the samples. The sodium benzoate and spice‐treated samples were all accepted by the panelist up to 60 days of storage with differing levels of acceptability (p ˂ .05); the untreated samples were not recommended at day 60 of storage, with a score of 2.83 ± 1.01.Sensory attributes of the treated and untreated cricket flour are shown in Table p ˂ .05). Additionally, males scored the color of sodium benzoate‐ and garlic‐preserved cricket flour substantially higher than females at day 0 and 60 of storage (p ˂ .05), respectively. Male and female panelists significantly differed (p ˂ 0.05), in their opinions of the aroma after 30 days of storage of cricket flour preserved with a combination of ginger and garlic extracts, as well as after 60 days of storage of sodium benzoate preserved cricket flour. The texture and overall acceptability ratings of the treated and untreated cricket flour samples did not significantly vary between the genders during storage. However, after 60 days of storage of the sodium benzoate preserved samples, the average overall acceptability of the male panelists (3.47 ± 0.92) was statistically higher than the female panelists (2.92 ± 0.82).Table There were strong negative correlations seen between the moisture content, pH, and the sensory qualities, while strong positive correlations were seen between the sensory attributes and overall acceptability Table . The eig44.1L* (lightness) was greatest on day 0 and lowest on day 60 in storage. Similarly, a* was highest on day 0 and lowest on day 30, though having a non‐significant difference among samples and storage duration, and the samples' tendency to yellowness (b*) declined over the course of storage, with no significant variation between samples. This could show the impact of long‐term storage on flour color. Cricket flour had lower lightness values than reported for other kinds of flours  was highest in untreated flour samples on day 60 (11.71 ± 0.55) and lowest in garlic (8.59 ± 1.06) and sodium benzoates (8.86 ± 0.35) preserved flour. This indicates the potential of the garlic and sodium benzoate to reduce chemical reactions taking place in food as a result of the interaction of nutrients in the flour with the environment. Deterioration of color during storage may also indicate a loss of nutritional content as a result of the auto‐oxidation reactions  flour  flour. At the end of 12 weeks of storage, TVC of ˂6 log cfu/g was reported in cricket (A. domesticus) powder, while yeast and molds were not detected , which was sufficient to destroy the fecal coliforms during the flour preparation, coupled with hygienic preparation processes. Live crickets are associated with a variety of pathogenic organisms, including fecal coliforms and E. coli  at the end of the 60‐day storage period. This was linked to the activity of the garlic antimicrobial compounds such as allicin which is reported to be stronger than ginger in previous studies , pH, and moisture content of cricket flour increased with the length of storage while the microbial load decreased during storage. When compared, the untreated cricket flour had a higher total microbial load, and yeast and molds compared to the treated, with garlic extract preserved flour having the lowest microbial load. Consumers liked both the spice‐treated and control cricket flour samples, but acceptability significantly declined with storage time. A strong negative correlation existed between the ΔE, pH, and moisture content of the cricket flour and the sensory score, while there was a significant positive correlation between sensory scores and overall acceptability. The study showed that using ginger and garlic extracts to treat crickets results in flour that is safe, shelf‐stable, and liked by customers. However, the sensory score and overall acceptance decline with time spent in storage. Therefore, the study concluded that treating crickets with ginger and garlic extracts produces flour that is safe, shelf‐stable and liked by consumers, although sensory score and acceptability are decreased with prolonged storage. Further research is recommended on nutrient, bioactives, anti‐nutrients, composition of spice treated cricket flour to promote its use in the food industry.The color change (ΔThis work was supported by the German Academic Exchange Services (DAAD) through the Regional Universities Forum for Capacity Building in Agriculture (RUFORUM), In‐Country/In‐Region Scholarship Programme #1 under Grant number 1D:57429563; International Foundation for Science (IFS) #2 under Grant number I‐3‐E‐6597‐1.The authors report there are no competing interests to declare.This study does not involve any human or animal testing.Data S1: Supporting informationClick here for additional data file.


Dalbergia sissoo, an economically important timber tree. In this study, we investigated the role of three differentially expressed genes identified in the dieback-induced transcriptome in Dalbergia sissoo. The transcriptome was probed using DOP-rtPCR analysis. The identified RGAs were characterized in silico as the contributors of disease resistance that switch on under dieback stress. Their predicted fingerprints revealed involvement in stress response. Ds-DbRCaG-02-Rga.a, Ds-DbRCaG-04-Rga.b, and Ds-DbRCaG-06-Rga.c showed structural homology with the Transthyretin-52 domain, EAL associated YkuI_C domain, and Src homology-3 domain respectively, which are the attributes of signaling proteins possessing a role in regulating immune responses in plants. Based on in-silico structural and functional characterization, they were predicted to have a role in immune response regulation in D. sissoo.Plant immunity includes enemy recognition, signal transduction, and defensive response against pathogens. We experimented to identify the genes that contribute resistance against dieback disease to  This tree has excellent value in forestry, agroforestry, horticulture, medicine, and the wood industry  are the putative or candidate R genes featured for their conserved structure and are involved explicitly in pathogen resistance to plants. They include NBS-LRR, receptor-like proteins, and kinases. The best-known class is NBS-LRR, classified based on the presence and absence of the Toll/Interleukin-1 Receptor (TIR) domain at the N-terminus. The non-TIRs are attributed to the coiled-coil (CC) domain and the less common leucine zipper (LZ) domain . The NBSthaliana , Oryza sa sativa , Medicaguncatula , Populuschocarpa , Vitis vvinifera , Cucumis sativus , Brassicica rapa , Carica a papaya , Sorghum bicolor , Brachypstachyon , and ZeaZea mays . The pre22.1R genes was used to probe the transcriptome of D. sissoo to identify the resistance gene sequence(s) against dieback disease. The NBS region of the NBS-LRR domain consists of conserved domains, such as kinase P-loop, kinase-2, kinase-3A, and hydrophobic GLPL motifs. Based on the NBS domain, degenerate primers were designed and synthesized to amplify resistance gene analogs (RGAs) against the dieback disease of D. sissoo (The NBS region of the NBS-LRR (Nucleotide Binding Site Leucine-Rich Repeats) class of . sissoo Table S12.2D. sissoo. Branch cuttings of healthy plants were taken and macropropagated in the greenhouse of the Fungal Molecular Biology Laboratory, Department of Plant Pathology, University of Agriculture Faisalabad, Pakistan. For nursery raising, the cuttings were planted in polythene bags filled with a mixture of soil and sand (3:1 ratio)  .2.3D. sissoo was inoculated with Ceartocystis dalbergicans  3 months post-inoculation. The plants that still showed no symptoms after 6 months were tagged as resistant sources and probed for identifying resistance genes.Macropropagated tagged material of ergicans . Spore r2.4Plants showing resistance under the dieback challenge were subjected to RNA isolation. The sampled leaves were immediately kept in liquid nitrogen prior to the RNA isolation procedure. Total RNA was isolated using the GeneJET Plant RNA Purification Mini Kit  following the manufacturer’s protocol. The dried pellet was dissolved in nuclease-free water and immediately used for downstream applications.2.5D. sissoo under dieback stress. The reaction conditions for the DOP-rtPCR were optimized. Amplicons were resolved on 1.2% agarose gel and visualized under UV on the gel documentation system . The required bands were cut and eluted from gel using a gel purification kit (FavorPrep) and sequenced on the Sanger platform . The generated sequences were characterized in silico. The generated sequences of identified RGAs were translated using the Expert Protein Analysis System (ExPASy) translate tool.The extracted RNA of each sample was quantified on a UV-visible NANODROP , and quality was assessed through gel electrophoresis. The RNA samples were subjected to cDNA synthesis. First-strand cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit , following the manufacturer’s protocol. Degenerate oligonucleotide primed-reverse transcriptase PCR (DOP-rtPCR) was performed to amplify putative disease resistance genes or RGAs  from the transcriptome of 2.6https://prosite.expasy.org/scanprosite/) was used for this purpose.The translated proteins were scanned for structural motifs from the PROSITE collection of motifs. The ScanProsite web tool from ExPASy   and PSIP3D. sissoo germplasm , theoretical pI (isoionic point), aliphatic index, instability index (II), and the grand average of hydropathicity (GRAVY) of the deduced amino acids  fingerprint. The protein synthesis elongation factor (EF-Tu) binds to the GTP molecule during the translation process and is now thought to be involved in plant stress response. The ELONGATNFCT is a five-element fingerprint that dispenses a signature to GTP-binding elongation factors. These five motifs were derived from the C-terminal site of the initial alignment of 12 elongation factors. The translated protein sequence of Ds-DbRCaG-01-Rga.a had shown homology with motif-2 and motif-4 in the ELONGATNFCT signature.The translated proteins of the 2+ channel gamma 6) fingerprint. Calcium ion (Ca2+) concentration is an essential indicator of signal transduction during plant growth, development, and stress. These consist of four subunits: beta subunit (intracellular); gamma subunit (transmembrane); the cleaved product of the same gene, alpha-2; and delta subunit , annotation grid or sequence plot, and PSIPRED chart were also computed (The SOPMA and PSIPRED programs were used for secondary structure assessments. The α-helix (Hh), random coil (Cc), and extended strand (Ee) percentiles were observed in the translated protein sequence of 3.5D. sissoo RGAs. The deduced amino acid sequences of the identified RGAs were subjected to SWISS-MODEL and PHYRE2 for template selection.Different online servers were used for the structural and functional characterization of the Caenorhabditis elegans). Twenty-three amino acid residues of Ds-DbRCaG-02-Rga.a (11-33) were modeled with template 3UAF residues (48-77) with 53.4% confidence and 39% sequence identity. The aligned region of the template confirmed the TTR52 superfamily (Transthyretin-like family) domain.The Ds-DbRCaG-02-Rga.a showed maximum homology with a significant hit to template 3UAF . Forty-six amino acid residues of Ds-DbRCaG-04-Rga.b (57-102) were modeled with template 2W27 residues (306-348) with 29.6% confidence and 21% sequence identity. However, the aligned region of the template confirmed EAL associated YkuI_C domain. This EAL domain is named after the presence of a conserved motif signature, Glutamate-Alanine-Leucine.The Ds-DbRCaG-04-Rga.b showed homology with a significant hit to the template 2W27 . Twenty-six amino acid residues (30-55) of Ds-DbRCaG-06-Rga.c were modeled with template 3M1U residues (135-160) with 62.6% confidence and 42% sequence identity. However, the template’s aligned region confirmed the SH3 (Src homology 3) domain, which involves regulating kinase activity.The Ds-DbRCaG-06-Rga.c showed homology with a significant hit to the template 3M1U  from 3.6After template selection, 3D imaging of these models was generated using PyMOL v. 2.4.0 Figure 23.7The quality was assessed by generating Ramachandran plots to confirm the reliability of homology-based generated 3D models. The model validation of Ds-DbRCaG-02-Rga.a predicted 82.4% residues in the most favored region and 11.8% in the additional allowed region with the remainder in the disallowed region. The model validation study of Ds-DbRCaG-04-Rga.b indicated 78.0% residues in the most favored region, 14.6% in the additional allowed region, and 7.3% in the generously allowed region. Similarly, a model validation study of Ds-DbRCaG-06-Rga.c indicated 83.3% residues in the most favored region, 11.1% in the additional allowed region, and 5.6% in the generously allowed region. Moreover, the calculated Z-scores for the predicted models of Ds-DbRCaG-02-Rga.a, Ds-DbRCaG-04-Rga.b, and Ds-DbRCaG-06-Rga.c were -0.16, -2.16, and -1.72, respectively, confirming the models to be satisfactory Figure 34D. sissoo RGAs were sequenced and characterized in-silico using a computational biology approach that proved part of the disease-resistance pathway. Subcellular localization confirms their location in the nuclear, mitochondrial, plasma membrane, and extracellular space. The extracellular location was also assessed with tobacco and Arabidopsis (NBS-LRR (class of R genes) based DOP-rtPCR analysis was performed to recover putative RGAs against dieback stress. In earlier studies, several plant species have been used in degenerate primers-based approaches to identify RGAs . These abidopsis . The dynbidopsis  which debidopsis  and NPR1bidopsis .D. sissoo were submitted. It confirmed their role as a subcellular entity; among these, the extracellular location Ds-DbRCaG-02-Rga.a and Ds-DbRCaG-06-Rga.c . This predicted role is in accordance with the pathogen-related proteins  of Nicotiana tabaccum , tryptophan (1.6-1.8%), and aspartate (1.6-3.3%), while the higher abundance of leucine (10.5-15.3%), proline (10.5-13.3%), serine (11.9-15.0%) and arginine (8.3-16.7%) in the deduced amino acid sequences was similar to the result as described by D. sissoo RGAs, when scanned for motif identification, displayed a protein kinase phosphorylation site that was observed in recovered RGAs. Various signaling pathways, including stress responses, mainly depend on protein kinases and phosphatases for phosphorylation or dephosphorylation . The protein synthesis elongation factor (EF-Tu) binds to the GTP molecule during the translation process and is now thought to be involved in plant stress response. EF-Tu and EF-1α were associated with high-temperature stress (The translated sequence of e stress .2+ channel gamma 6) fingerprint. Calcium ion (Ca2+) concentration is an essential indicator of signal transduction during plant growth, development, and stress. Voltage-dependent Ca2+ channels in the plasma membrane are responsible for Ca2+ influx and are activated upon ionic imbalance depolarization during hormonal, environmental, or pathogenic stimuli  and phytochelatins (PCs) proteins are involved in metal detoxification and also in stress response in plants . During in-silico characterization of D. sissoo RGAs was performed using online web servers. Ds-DbRCaG-02-Rga.a showed significant homology with template PDB ID: 3UAF. The alignment region confirmed TTR52 (Transthyretin-52) domain acts as a bridging molecule for phagocytes and apoptotic cells, thus regulating immune responses, previously shown to regulate cold stress in Arabidopsis  domain. It acts as a bridging molecule for phagocytes and apoptotic cells, thus regulating immune responses . In addiDs-DbRCaG-04-Rga.b showed homology with a significant hit to the template 2W27, and the aligned region of the template confirmed the EAL-associated YkuI_C domain. This EAL domain is named after the presence of a conserved motif signature, Glutamate-Alanine-Leucine. It possesses type-I cyclic-dimeric-GMP specific phosphodiesterase (PDE) activity and has been identified as a signaling protein that plays a role in infection  domain, which regulates kinase activity. The SH3 domain works as a polypeptide cofactor for activating or inhibiting different kinases (The  kinases . The SH3The original contributions presented in the study are included in the article/SI and IU conceived, designed, and developed the experiment. SI, IU, HR, and BN experimented. SI and IU analyzed and interpreted the data. SI, IU, HR, BN, HA, and SK drafted and revised the manuscript. All authors contributed to the article and approved the submitted version.