The long interspersed element 1 (LINE-1 or L1) integration is affected by many cellular factors through various mechanisms. Some of these factors are required for L1 amplification, while others either suppress or enhance specific steps during L1 propagation. Previously, TRIM28 has been identified to suppress transposable elements, including L1 expression via its canonical role in chromatin remodeling. Here, we report that TRIM28 through its B box domain increases L1 retrotransposition and facilitates shorter cDNA and L1 insert generation in cultured cells. Consistent with the latter, we observe that tumor specific L1 inserts are shorter in endometrial, ovarian, and prostate tumors with higher TRIM28 mRNA expression than in those with lower TRIM28 expression. We determine that three amino acids in the B box domain that are involved in TRIM28 multimerization are critical for its effect on both L1 retrotransposition and cDNA synthesis. We provide evidence that B boxes from the other two members in the Class VI TRIM proteins, TRIM24 and TRIM33, also increase L1 retrotransposition. Our findings could lead to a better understanding of the host/L1 evolutionary arms race in the germline and their interplay during tumorigenesis. The L1 fication . Althougfication . As is tfication . Specifiin trans .Retrotransposition events were initially detected in the germline, and during early embryogenesis . Some ofTripartite motif-containing (TRIM) family proteins are crucial members in the innate immune response and have a broad effect on restricting viruses and retroelements ,49. It hTRIM28 gene, also known as KAP1 (Krüppel-Associated Box (KRAB)-Associated Protein 1) or TIF1-β , encodes for TRIM28 protein, a member of the Class VI TRIM family proteins, which share many structural characteristics . TRIM28 de novo L1 inserts in transiently transfected cultured cells and by analysis of de novo tumor-specific L1 inserts in tumors of patients with endometrial cancer, prostate cancer, and ovarian cancer. Together, our studies have identified previously unknown effects of TRIM28 on potentially distinct steps in L1 retrotransposition cycle and provided patient-based data supporting in vivo relevance of our findings.In this study using previously reported ORF2p fragments ,34,78 anPlasmids containing DNA encoding human and mouse TRIM28 were obtained from Origene  and subcloned into a PCDNA 3.1/Hygro+ vector using NheI and HindIII restriction enzymes. The coding sequences for fragments of human TRIM28 were synthesized by Genescript, including RBCCH (AA 1–509), RBB (AA 1–244), RING (AA 1–139), BB (AA 139–244), CC (AA 244–405), N-P-Br (AA 509–835), N-P AA 509–680), P-Br (AA 617–835), NHD (AA 509–617), PHD (AA 617–680), Br (AA 680–835). The break points for TRIM28 domains were used as described ,68. The 0, P-Br , includiThe ORF2 constructs were generated as previously described ,34,78. SHeLa cells were maintained as previously described . U2OS ceThe U2OS TRIM28 KO cell line was generated using CRISPR-Cas9 gene-editing using the protocol as previously described . Briefly500 000 cells were seeded 16–18 h prior to transfection in T75 flasks. 0.2 ug L1Neo or 0.6ug L1 Blast expression plasmid  and 0.8 For the L1 retrotransposition assay in Figure Five hundred thousand HeLa cells or U2OS cells were seeded in T75 flasks. Cells were transfected next day using Lipofectamine reagent (Life Technologies) following the manufacturer's protocol with 0.1 μg of pIRES2-EGFP and 0.8ug of indicated constructs. The plasmids were mixed with 12 ul of Plus reagent (Life Technologies) in 180 ul serum free media. Then 85 ul of serum free media was mixed with 15 ul Lipofectamine reagent (Life Technologies) before being added into the previous DNA mix. The transfection cocktail was added to the cells in 6 ml of serum free media for 3 h, after which it was replaced with 9 ml of Minimum Essential Media (MEM). Cells were maintained under selective medium containing 500μg/ml G418 (Life Technologies) for 14 days starting 48 h post transfection before being fixed and stained with crystal violet solution . Selective medium was changed every 3 days. The number of Neomycin-resistant colonies were counted in each flask. Statistical analysis was performed using unpaired two sample t test compared with control.Adapted from ,83, two Cell lysates were either treated (+) or not treated (−) with 5μl RNase A  and transferred to nitrocellulose membranes using the iBlot2 system (Life Technologies). The XCell Blot Module was used for wet-transfer module for ORF2p analysis. Membranes were blocked in PBS-Tween  with 5% blotting-grade blocker (BioRad) and incubated with primary antibodies overnight at 4°C. Targeted proteins were detected with corresponding primary antibodies: hORF1  antibody; abcam anti-T7 tag ab9115; cell signaling anti-FLAG #2368S; cell signaling TIF1β Antibody #4123; cell signaling GAPDH (14C10) Rabbit mAb #2118. Membranes were washed 3× in PBS–Tween for 5 min before incubation with secondary antibodies. Secondary antibody HRP-mouse anti-rabbit (Santa Cruz: sc2357) or HRP-mouse anti-goat (Santa Cruz: sc2354) was applied for 1 h at room temperature (1:5000 in 3% blotting grade blocker/PBS–Tween). Images were captured using a BioRad Gel Doc XR+ imager. GAPDH was used as loading control.2HPO4 (Sigma S3264) and 2 mM KH2PO4 (Sigma P9791), pH  7.4) and harvested using 500 μl of TLB , pH  7.2) buffer per T75 flask. The samples were centrifuged at 13 000 rpm for 15 min at 4°C. The supernatant was transferred to a new microcentrifuge tube (this is the cytoplasmic fraction). The remaining nuclear pellet in the microcentrifuge was gently washed three times with 200 ul of TLB buffer. The nuclear pellets were resuspended in TLB and the nuclear lysate samples were sonicated with a Microson XL-2000 sonicator (Misonix) 4× (6 s sonication/10s rest on ice). Samples were centrifuged at 21 130g for 15 min at 4°C. The resulting supernatant (the nuclear fraction) was then transferred to a new microcentrifuge tube. The protein concentration for each sample was determined using 595 nm wavelength OD values against a bovine serum albumin (BSA) standard.The processing of nuclear and cytoplasmic fractions was performed as previously described ,84 SpeciFor each transfection, 50 000 cells were seeded onto each glass slide. The cells were transfected with 1ug of each indicated plasmid and fixed and stained 24 h post transfection. Primary antibodies were incubated for overnight at 4°C  were incubated for 1 h at room temperature. Slides were mounted with DAPI , and confocal analysis was performed using Nikon Ti2 confocal microscope.Briefly, 4 million HeLa cells were seeded 16–18 h prior to transfection in T75 flasks and co-transfected with 5 ug of each indicated plasmid. 12 ul of Plus reagent (Life Technologies) in 180 ul serum free media was mixed with 24 ul Lipofectamine reagent and 76 ul serum free media (Life Technologies). The transfection cocktail was added to the cells in 6 ml of serum free media for 3 h, after which it was replaced with 10 ml of Minimum Essential Media (MEM). Approximately 48 h post-transfection, cells were washed as described above and harvested in 5 ml PBS. Cells were pelleted at 3000g for 5 min at 4°C and then lysed in 500 l of LEAP lysis buffer . After incubation on ice for 5 min, samples were centrifuged for 5 min at 3000g at 4°C to remove debris. Supernatant was layered on top of an 8.5% sucrose cushion that was layered on top of a 17% sucrose cushion, and then centrifuged at 36 500 rpm for 2 h at 4°C. Supernatant was removed, and pellet was resuspended in 100 ul deionized water supplemented with HALT protease inhibitor and RNAsin. Protein concentration was assessed using Bradford assay and samples were brought to equal protein concentrations. Samples were stored at –80°C in final 50% volume/volume glycerol. ORF2p-mediated LEAP reaction was performed using 1ug of each sample. The following primers were used to assess ORF2p-generated cDNA:LEAP forward primer 1: AAGGACACCTGCACCCGGATGTTCATCLEAP forward primer 2: TCCTGCGGGACCTGGAGCTGGAGATLEAP forward primer 3: CATCATCAAGAAGAGCGGCAACAACLEAP forward primer 4: CTTCAGCAAGGAGGACATCTACGCLEAP forward primer 5: AAGATTTTCGCCACCTACAGCAGCGACAAGLEAP reverse primer: AGTGGCACCTTCCAGGGTCAAGGAAReverse Transcriptase System (Promega: A3500) was used according to manufacturer's instructions. Reactions were set up at 9 ul sample. Each sample was done in duplicates with one for RT+ one for RT–. The RT+ reaction was added with 1.5 ul of AMV, the RT- reaction was added with distilled water. The final volume was brought up to 20 ul with distilled water. The following samples were incubated at 70°C for 10 min. For each sample, the same set of primers in LEAP assay were used for conventional RT-PCR.G418 resistant clones were selected as described for retrotransposition assay. Individual Neo-resistant colonies were picked and expanded in 12 well plates in 500 μg/ml G418. Genomic DNA was extracted from cells generated from individual colonies. Thirty-two clones were randomly selected from each treatment and subjected to genomic DNA extraction. GoTaq-Hot start polymerase (Promega PRM5122) was used for the PCR reactions. Each PCR reaction contained 100–200 ng of genomic DNA, 2 ul of each 10uM primer, and nuclease-free water was used to bring up the total volume of 50 μl. Typical amplification conditions were: 95°C, 3 min;  × 35 cycles; 72°C, 5 min; 4°C hold on thermocycler. Oligonucleotides used for PCR included the following: reverse primer: 5’-ATTGAACAAGATGGATTGCACGC-3’; forward primer: 5’-CAGGGATGCCCTCTCTCACCG-3’. PCR products were analyzed on 1% agarose gels.de novo insertions. In brief, the detected L1 insertions in tumor and blood/normal tissues were defined as true positive calls with the filter label ‘pass’. All the true positive L1 insertions found in tumor samples were cross-referenced with the ones detected in blood/normal tissues. Only the ones not detected in blood/normal samples but unique to tumor samples were considered as tumor specific L1 insertions. Tumor specific full-length L1 insertions were cross referenced with a list of reported polymorph L1s  on polyA-selected cytoplasmic RNA was performed by BGI as previously described . RNA-SeqP value . These dFor gene expression normalization, GraphPad Prism 9.4 was used for analysis. All genes across 12 different samples  were normalized between 0 and 100%. Zero was defined as the smallest value in each data set, and one hundred was defined as the largest value in each data set. All values in between were then normalized accordingly .Previous studies have oppositely shown that TRIM28 acts as either a potentially positive or negative regulator of L1 retrotransposition in HeLa-M2 cells and K562 cells ,58. SincTo determine whether this effect can be observed in other cell lines and to determine whether TRIM28 is required for L1 retrotransposition, we generated TRIM28 knock out U2OS cells (U2OS KO)  that do not express endogenous TRIM28 but support equal expression levels of transiently transfected human TRIM28 protein Figure . L1 retrOne possible mechanism through which TRIM28 may promote L1 retrotransposition is through interaction with L1 proteins. To test whether human and mouse TRIM28 proteins interact with human L1 ORF1 or ORF2 protein , we transiently co-expressed FLAG-tagged H-TRIM28 or M-TRIM28 with L1 ORF1p or T7-tagged L1 ORF2p and performed co-immunoprecipitation in HeLa cells using beads conjugated with anti-FLAG antibodies Figure  but not To identify the domains of the human ORF2p responsible for the interaction with TRIM28, we generated a series of T7 tagged ORF2p fragments . Plasmids containing each of these fragments were individually transiently co-transfected with a plasmid expressing either a full-length H-TRIM28-FLAG or a full-length M-TRIM28-FLAG proteins. Cellular lysates collected from HeLa cells co-transfected with these combinations of plasmids were used to perform co-immunoprecipitation followed by western blot analysis . This apTo identify whether a specific part(s) of the human TRIM28 may be responsible for increasing retrotransposition of the Neo-tagged L1, we generated plasmids containing different domains of human TRIM28 protein as previously described ,68 Figu. PlasmidTo determine whether the effect of TRIM28 fragments on L1Neo retrotransposition is dependent or independent of their ability to interact with L1 ORF2p, we performed a series of Co-IP experiments in HeLa cells using TRIM28 fragments and the ZRT fragment of human ORF2p. The ZRT construct is chosen to screen these interactions because the ZRT protein fragment contains an important enzymatic activity of the ORF2p and is easily detectable compared to other ORF2p fragments and the full-length L1 ORF2p. Western blot analysis of co-IP lysates demonstrates that all TRIM28 N- and C-terminal domains can interact with ZRT to a certain extent . In addiTo further characterize the TRIM28-ZRT interaction we evaluated subcellular localization of this interaction using western blot analysis. HeLa cells transfected with the TRIM28 and ZRT expression plasmids separately or together were fractionated to separate nuclear and cytoplasmic parts as previously described ,84. EachIt is reported that the higher-order assembly of TRIM28 dimers is dependent on B box interactions, which could be abolished by three characterized mutations A160D/T163A/E175R . To inveConsistent with our findings with the triple mutant B box, full-length triple mutant TRIM28 (TRIM28 3M) is unable to increase L1 retrotransposition compared to TRIM28 WT Figure , D. WestN = 4 for each condition). This time point was chosen based on previous findings that L1 retrotransposition in this assay is detectable after 36 h post-transfection (P < 0.05). We used RNA-Seq by Expectation-Maximization (RSEM) analysis and gene set enrichment analysis (GSEA) to detect cellular pathways altered in the TRIM28 WT samples compared to the Control and TRIM28 3M samples (N = 4 each). This approach also identified DNA repair as a significantly downregulated pathway when TRIM28 WT was overexpressed   Figure   Figure . Based o) Figure . These rOur results show that TRIM28 protein interacts with L1 ORF2p but not ORF1p Figure  support in vitro can be analyzed using the well-established L1 element amplification protocol (LEAP) (The RT activity of ORF2p l (LEAP) ,86,99. Il (LEAP) ,78. To al (LEAP) . This apl (LEAP) . To confl (LEAP) . This rel (LEAP) . Additiol (LEAP) . As expel (LEAP) . This prl (LEAP) . In paral (LEAP) . This fiP = 0.04). Although no significant differences were detected in TRIM28 WT versus 3M (P = 0.2), the 3M group was also not different from the Control group and there is a trend of fewer 3 kb positive clones being formed when TRIM28 WT is overexpressed compared to the 3M construct . Consistent with our findings with the LEAP assay, TRIM28 WT overexpression leads to shorter de novo L1Neo inserts, compared to the control or TRIM28 3M overexpression.To confirm that our LEAP results, which are obtained using cytoplasmic RNPs, are relevant to L1 retrotransposition products, we performed a ruler PCR assay with the reported 3 kb primers . This siin vivo, we identified patients with endometrial cancer, prostate cancer, and ovarian cancer using GDC Legacy database. Patients within each cancer group were separated in to two groups, with 8 patients demonstrating ‘High’ and 8 patients exhibiting ‘Low’ TRIM28 mRNA expression according to the reported TRIM28 mRNA expression in their tumors , and so do the patients with prostate cancer  and ovarian cancer . All patDe novo tumor specific L1 insertions were identified as present in the tumor WGS but not in blood/normal WGS and meeting MELT criteria of de novo L1 inserts as described . Based on the described inclusion criteria, we could not identify sufficient number of patients per group to conclusively determine whether TRIM28 expression levels affect the number of de novo L1 inserts per patient. However, the large number of total de novo L1 inserts identified in each group of patients has allowed us to compare their lengths. Unpaired two-sample t test analysis determined that de novo L1 insertions are significantly shorter in the ‘High’ TRIM28 expression group compared to the ‘Low’ TRIM28 expression group of patients in each cancer cohort . These values are calculated using all tumor-specific L1 inserts, which include those that may have happened before the increase in the TRIM28 expression occurred in any given tumor. The number of full-length L1 inserts in each group was too small to analyze them as a separate category. These patient-based findings agree with our findings in HeLa cells using LEAP assays  or binds to the RNA template which facilitates pausing or dissociation of the whole complex from the template RNA. Second, TRIM28 directly or indirectly binds to the mRNA template and interferes with ORF2p reverse transcription by blocking its forward progress, which ultimately leads to ORF2p pausing and/or dissociation from the RNA template. These models are consistent with the reported ability of TRIM28 to interact with both proteins and nucleic acids  and invoin vivo. TRIM28 is ubiquitously expressed throughout development and in adult tissues. In embryonic stem cells the expression level of L1 and TRIM28 are both high (de novo L1 integration is beneficial to driving cancer evolution regardless of the size of L1 inserts. The reverse order of these evolutionary steps is also possible. According to this hypothesis, L1 may have evolved to use TRIM28 to increase the number of its inserts. To counteract this the host utilized the same mechanism to reduce the number of full-length L1 inserts to halt L1 propagation.Our findings of the effect of TRIM28 on L1 retrotransposition and cDNA synthesis in different cancer models may have important biological implications for understanding evolutionary arms race between the host and L1. Most of the 500 000 L1 elements in the human genome are 5′ truncated , but theoth high ,132. It In summary, we have identified TRIM28 as a suppressor of ORF2p-mediated cDNA synthesis, but overall, a positive regulator of L1 retrotransposition. Both effects rely on the B Box facilitated oligomerization. These findings have evolutionary and disease relevant consequences that may be related to other viral and non-viral RT containing proteins. Although TRIM28 is rarely mutated in human cancers, its expression is often deregulated with overexpression being linked to poor survival. Our findings support that the ability of TRIM28 to promote tumorigenesis may include its potential to increase L1-induced genomic instability. Future mechanistic studies of TRIM and L1 proteins will be needed to provide a more comprehensive understanding of functional outcomes of their interactions on genome stability, evolution, and cancer pathogenesis.The raw data files for RNA-seq analysis in this manuscript are available under BioProject: PRJNA925462.gkad247_Supplemental_FilesClick here for additional data file.


This review mainly addresses applications of polymer/graphene nanocomposites in certain significant energy storage and conversion devices such as supercapacitors, Li-ion batteries, and fuel cells. Graphene has achieved an indispensable position among carbon nanomaterials owing to its inimitable structure and features. Graphene and its nanocomposites have been recognized for providing a high surface area, electron conductivity, capacitance, energy density, charge–discharge, cyclic stability, power conversion efficiency, and other advanced features in efficient energy devices. Furthermore, graphene-containing nanocomposites have superior microstructure, mechanical robustness, and heat constancy characteristics. Thus, this state-of-the-art article offers comprehensive coverage on designing, processing, and applying graphene-based nanoarchitectures in high-performance energy storage and conversion devices. Despite the essential features of graphene-derived nanocomposites, several challenges need to be overcome to attain advanced device performance. Progression in energy technologies demands the use of innovative competent nanomaterials to attain the desired high performance of those technologies . GraphenIn recent years, functionally graded graphene-reinforced composites have been focused on heavily in the literature . FunctioThis state-of-the-art review primarily covers the fundamentals of graphene and its nanocomposites. Additionally, the energy storage and conversion solicitations of graphene-derived nanocomposites are scrutinized in terms of important devices. Accordingly, high-efficiency multi-functional supercapacitors, batteries, and fuel cells are debated. To the best of our knowledge, this article is pioneering in the field of energy storage and conversion devices in terms of the review outline, the collected literature, and the wide coverage of various devices to study the effect of graphene-based nanocomposites. Few previous reviews of the literature on graphene materials have included a comprehensive survey in order to support future progress in this field . The fut2-hybridized hexagonal carbon atoms with π-orbitals [2V−1s−1 and 3000–5000 W/mK, respectively [Graphene is considered an excellent nanocarbon form ,23,24. Iorbitals ,26,27. Forbitals ,29,30. Sorbitals ,32,33,34orbitals ,36,37,38orbitals ,40. The ectively ,42. In aectively .Graphene nanosheets can be held together through van der Waals interactions ,45,46. W2-hybridized structure of graphene and styrene rings of polymer  features [−2. Jiao et al. [−1 and an efficiency of ~88% through 100 cycles. Hence, the effectiveness of using graphene in Li-ion battery anodes is established.Li-ion batteries have also been used as effective energy storage devices ,93. Prevfeatures . Li and features  designedo et al.  formed a−1. Moreover, the polyaniline-grafted graphene-oxide-derived anode had a high cycling stability and Coulombic efficiency. Guo and researchers [−1), and an extended cycling life . Chae et al. [−1 was attained. Hence, the inclusion of graphene and graphene oxide provided considerable benefits to the performance of the Li-ion battery due to dispersion and interactions with the compatible polymer structures [Conducting polymer and graphene-derived nanocomposite electrodes have been designed for Li-ion batteries . Li et aearchers  reinforce et al.  worked oructures ,107.In lithium-ion storage batteries, basically different graphene-based nanomaterials have been used for electrodes such as graphene-supported polymers, graphene-supported sulfides, graphene-supported metal oxides or alloys, etc. . The syn−2 and ~311 mA cm−2, respectively, relative to the three-dimensional graphene oxide . Moreover, the modified three-dimensional graphene oxide exhibited an elevated proton conductivity of 3.2 S cm−1 relative to the non-modified three-dimensional graphene oxide (0.7 S cm−1). The use of the three-dimensional graphene oxide with inserted sulfate ions was suggested to enhance the proton conduction capability of the nanomaterials due to their superior fuel cell performance. The fuel cell is an incipient energy conversion technological development employing nanocomposite structures for electrolyte membranes or catalysts ,112. ConLee et al.  fabricatTraditionally designed supercapacitors, batteries, or fuel cells have high price, high density, and low structural reliability problems . Before The commonly used nanocarbon nanofillers for device electrodes and components include graphene, graphite, carbon nanotube, etc. Among these, graphene has been found to be the most efficient to enhance the performance of various energy devices. Moreover, the use of conjugated polymers has been found to enhance the electron and charge transportation through these remarkable nanomaterials ,127. It More specifically, in supercapacitors, different graphene-derived nanocomposites have been utilized. Polymer/graphene nanocomposites with a high surface area and pseudocapacitance may result in a fabulous enhancement in the supercapacitor performance . ConsequAlthough promising results have been attained for numerous graphene-nanocomposite-derived Li-ion battery electrode materials, there are some prevailing challenges that need to be overcome for future developments . AccordiThe inclusion of graphene in nanocomposites may cause high performance and resilience for fuel-cell-based energy conversion devices . GraphenHenceforward, the future of graphene nanomaterials in energy devices greatly rely on the development of new innovative materials. In this context, using three-dimensional graphene-derived nanocomposites in energy systems may bring revolution in this field . The conFacile approaches need to be adopted to achieve efficient graphene-derived nanocomposites . High-peThis overview exclusively and comprehensively debates the progresses in the arena of graphene and graphene-derived nanocomposites, concentrating on the energy storage and conversion solicitations. Graphene-derived nanomaterials have been constantly researched for supercapacitance, Li-ion batteries, and fuel cell applications. These devices have been categorized among the most essential energy storage and conversion devices. The investigations exposed the stability, charge storage, charge capacity, and conversion efficiency of these energy systems while employing some efficient carbon nanomaterials. In the case of advanced supercapacitors and batteries, using graphene, graphene oxide, functional graphene, and related nanocomposite nanomaterials amended the charge storage performance as well as the power conversion and utilization capabilities of the devices. The employed polymers along with the graphene-based nanofillers were found to be important to develop the high-efficiency energy-associated devices and systems. In other words, the choice of graphene nano-reinforcements along with the selection of polymers were considered indispensable for a better performance of the energy devices. In addition, the graphene modification as well as the graphene nanocomposite processing methods have played imperative roles in the enhancement of the charge storage and energy-production-related performance of these materials, resulting in high-efficacy supercapacitors and other devices. Consequently, the indispensable features of the energy devices and systems were enhanced using graphene or its derived nanofillers, graphene synthesis methods, nanocomposite fabrication techniques, etc., leading to homogeneous microstructure, matrix–graphene interactions, electron conduction, specific capacitance, charge capacity, high charge storing capability, charge–discharge, life cycle, charge density, power density, cyclic concert, reliability, and numerous related features of these systems.Briefly speaking, in this article, we debated the topical research advancements in the graphene and graphene-derived nanocomposites for important energy storage and conversion systems. Although substantial developments have been made so far, the marvelous potential for real-world applications in cutting-edge energy systems still requires further research. Importantly, the fabrication of graphene and its related nanocomposites for diverse energy applications are still in premature stages, where gaps exist in understanding the atomic/molecular level functioning, essential limits, and failure of devices. Moreover, the high surface area of graphene-derived nanomaterials plays a significant part in supercapacitors and batteries for charge storage. The overall nanocomposite design supporting the combination of a high surface area and pseudocapacitance of graphene may result in a tremendous improvement in the charge-storing potential of devices. To overcome the challenges of energy storage systems, it is essential to develop and understand the multidisciplinary tactics and connections between materials science, engineering, microstructures, electrochemistry, physical features, and interactions.Research has revealed the potential of graphene nanocomposites towards the electrodes of rechargeable Li-ion batteries. The high-energy densities of Li-based materials were found to be favorable for electronic energy storage. Mostly, the anode systems in Li-ion batteries benefited from using graphene nanocomposites due to their high surface area and conductivity values. All of these factors caused the high power density and rate performance of the Li-ion battery anode. In this context, the high surface area of graphene can accommodate more Li-ions to improve the energy density and capacity of the batteries. Carefully designed graphene-nanocomposite-based electrodes have been applied for commercial-level applications. On the other hand, the high surface area, aggregation trends, and porosity of graphene-derived nanomaterials can also decrease the performance of the electrode materials due to the formation of the solid electrolyte interphase layer causing a low first cycle Coulombic efficiency. Hence, the overall practical applications of graphene nanocomposites in energy storage systems have been limited. Moreover, using porous graphene-based materials may absorb large amounts of electrolytes, leading to battery swelling during electrochemical processes and also cost issues.Moreover, unusually high physical/chemical properties of graphene nanocomposites have been used for fuel cell applications due to their efficient proton conduction. Graphene-nanocomposite-derived fuel cell catalysts have cost effectiveness, high durability, and insensitivity to carbon monoxide during electrocatalysis relative to platinum-based catalysts. In proton-conducting electrolytes, graphene derivatives have oxygen functionalities to hold water and develop the proton transportation channels. However, due to blending with polymers, some challenges may arise like aggregation, decreased surface area, and homogeneity of the electrolyte membranes. Hence, graphene has been blended with different polymers in order to fabricate nanocomposites for enhancing the processing, mechanical stability, chemical and electrochemical features, and other functions for applications in supercapacitors, batteries, and fuel cells. Although great evolutions have already been attained, the research in this field still faces several unresolved glitches.Hence, this overview portrays the developments in the field of graphene nanomaterials for energy storage and conversion. In this context, several graphene nanofillers and derived nanomaterials along with polymers such as conjugated polymers, thermoplastics, and thermosets have been used for nanocomposites in order to attain advanced features of the energy devices. Appropriately adopting graphene-derived nanomaterials offers some notable future opportunities towards important energy storage and conversion systems. Moreover, constant research efforts may resolve the challenges related to the design, properties, and performance of applying graphene materials in energy devices. Forthcoming research may extend the use of the above conversed graphene-based nanomaterials towards efficient photovoltaics, light-emitting diodes, nanogenerators, microelectronics, and numerous other devices.


Due to its outstanding properties, graphene has emerged as one of the most promising 2D materials in a large variety of research fields. Among the available fabrication protocols, chemical vapor deposition (CVD) enables the production of high quality single-layered large area graphene. To better understand the kinetics of CVD graphene growth, multiscale modeling approaches are sought after. Although a variety of models have been developed to study the growth mechanism, prior studies are either limited to very small systems, are forced to simplify the model to eliminate the fast process, or they simplify reactions. While it is possible to rationalize these approximations, it is important to note that they have non-trivial consequences on the overall growth of graphene. Therefore, a comprehensive understanding of the kinetics of graphene growth in CVD remains a challenge. Here, we introduce a kinetic Monte Carlo protocol that permits, for the first time, the representation of relevant reactions on the atomic scale, without additional approximations, while still reaching very long time and length scales of the simulation of graphene growth. The quantum-mechanics-based multiscale model, which links kinetic Monte Carlo growth processes with the rates of occurring chemical reactions, calculated from first principles makes it possible to investigate the contributions of the most important species in graphene growth. It permits the proper investigation of the role of carbon and its dimer in the growth process, thus indicating the carbon dimer to be the dominant species. The consideration of hydrogenation and dehydrogenation reactions enables us to correlate the quality of the material grown within the CVD control parameters and to demonstrate an important role of these reactions in the quality of the grown graphene in terms of its surface roughness, hydrogenation sites, and vacancy defects. The model developed is capable of providing additional insights to control the graphene growth mechanism on Cu(111), which may guide further experimental and theoretical developments. Graphene, as the first isolated two-dimensional 2D) material , has attD materiaThe CVD process is highly dependent on the synthetic parameters, such as the partial pressure of precursors, temperature, substrate surface, and carbon solubility in the metal substrate ,36,37. A domains ,41,42,43 domains . Moreove domains ,46, therThe controllable synthesis of large-sized graphene using CVD is still a challenge due to the complex growth process and its sensitivity to growth conditions . A detaiThe mechanism of graphene growth in CVD has a multiscale character, i.e., it requires multiscale modeling approaches to properly complement experimental investigations. Numerous theoretical studies, employing ab initio calculations , moleculWhen combined with first principle calculations, the KMC method emerges as a promising approach that is capable of characterizing graphene growth with atomistic resolution and for significantly larger time and length scales compared to other models. Such multiscale models were developed by, e.g., Li et al. , who usen et al.  proposedu et al. , who shoan 10 nm ,60,61,62an 10 nm  developeDespite all these outstanding efforts to model the growth of graphene for isolated flakes, important questions, concerning the impact of the kinetic pathways on the atomistic scale of reactions involved, remain open. Developing KMC methods that are capable of providing insight into the atomistic scale requires consideration of a complete list of reactions, which was previously limited to the size of the simulated graphene flakes ,60,61. FIn this paper, we report a DFT-based KMC model capable of dealing with a comprehensive list of reactions, ranging from the dissociative adsorption of CHIn order to analyze the growth mechanism of graphene, we utilized the density functional theory (DFT) method to determine the activation energy barriers for relevant possible reactions involving the species of interest, i.e., H, C, CH, CHSince Li et al. reported activation energy barriers of the set of reactions happening during the CVD of graphene on Cu(111) using CHecursors , we firsecursors . In parti et al. . TherefoThe BEEF-vdW functional makes it possible to determine the barrier height for reactions on surfaces very accurately, since it was especially designed for the processes on surfaces . At the We found a reasonable agreement between the results obtained by the PBE-D3 and PBE-D2 , but a mi et al. . This wai et al. ). TherefUsing the activation energy barriers and reaction energies obtained, we calculated the linear scaling relations for the detachment of “small species” from the graphene edges . We alsoFinally, we have to mention that we also calculated the hydrogenation and dehydrogenation reactions, which were missing in the previously reported approach. We have added these reactions to the list of reactions used in the KMC method see . They arIn order to study the mechanism of graphene growth on a Cu(111) surface, we utilized the KMC protocol with reaction rates obtained from the DFT as parameters. Eight different simulations were carried out to examine the impact of partial pressures on the rates of chemical reactions occurring during the growth process see .Each simulation resulted in a non-equilibrium steady-state being reached at approximately on plots . In the ions see . Conversions see .ver time . After atate see . This petate see b. Since tate see c,d.We conducted a comprehensive analysis of the spatio-temporal evolution of all the samples refer to . Our fin quality ,39,42,72time see . It is cFinally, we observed a high concentration of H adatoms  in detail. The precursor used in this study is CHtion see  and the tion see . Therefocess see .Moving on to the next set of reactions, CThe contribution of edge attachments of carbon and its dimer were found to be the most stable, while other attached species were more likely to be hydrogenated and detached, as indicated by the arrows in To successfully grow graphene, it is crucial to allow ring closure reactions, which are highlighted by the brown region in d growth . The incTo evaluate the impact of (de)hydrogenation on graphene growth, we conducted KMC simulations with and without the set of (de)hydrogenation reactions for samples S1 hydrogenation on the quality of the grown graphene is evident from the results. The surface roughness of the samples was substantially lower in the presence of (de)hydrogenation reactions, with values of 1.64 nm and 1.85 nm for S1 and S2, respectively, compared to 1.98 nm and 2.12 nm, respectively, when the reactions were absent. Additionally, the inclusion of (de)hydrogenation reactions led to a decrease in root-mean-square roughness, whose values were reduced by 0.27 nm and 0.36 nm for S1 and S2, respectively. To further analyze the roughness of the samples, we used the radius as a measure and calculated the roughness profile, which shows the deviation of the radius from the mean radius hydrogenation reactions, as demonstrated by the surface roughness analysis and the observed reduction in the number of hydrogenated edges and defects (vacancies). The results suggest that incorporating (de)hydrogenation reactions on the edges leads to higher quality graphene material.For the development of the multiscale model, we considered the CVD conditions depicted in reaction ,75; therreaction , we neglAll quantum mechanical calculations were performed using density functional theory with the BEEF-vdW  and PBE To calculate reactions between “small species”, e.g., the dehydrogenation of methane, the Cu(111) surface was modeled by a four-layer 3 × 3 slab (with 2 bottom layers being frozen), see  in Ref. .In the present study, we have not considered subsurface processes. However, we included five reaction barriers for the formation and reconstruction of the hexagon at the graphene edge, which were proposed by Chen et al. . TransitCollection scheme, in which the algorithm traverses the lattice points to collect species and, after considering their neighboring sites and the reference reactions list, collects all possible reactions from current configuration as a list; (ii) a Selection scheme, where the algorithm calculates the cumulative  rate of all possible reactions collected in the previous step and generates two random numbers ; and (iii) an Update scheme with three stages, which include executing the selected reaction based on its type, advancing the simulation time using the second random number generated before, and updating the lattice configuration accordingly.To efficiently simulate CVD growth processes with elementary steps characterized by diverse rates, we developed an algorithm based on the rejection-free KMC approach, known as the BKL algorithm ,88. We aWe calculated all reaction rates, except for the CHry (TST)  using acximation  considerOne of the main aims of this study was to determine how the growth of graphene using CVD is affected by the partial pressures of CHTo study the CVD growth of graphene on a Cu(111) surface at 1300 K, we developed a multiscale model by combining first principle calculations and the KMC method. By implementing a kinetic Monte Carlo program, we were able to accurately represent relevant atomic scale reactions without any additional approximations, thereby allowing us to study long time and length scales. Specifically, we were able to observe graphene flakes of up to 38 nm that formed in 0.157 s under different CHrefer to . SpecifiWe also identified the role of individual reactions that occurred during graphene growth and studied the reaction pathways in detail. Data analysis suggested that the carbon monomer (C) and dimer , thus providing insights into how controlling parameters affect the quality of the material. This method is particularly helpful for understanding the kinetics of CVD graphene growth and other experimental observations at the relevant temperatures and pressures used in the production of graphene. However, it is important to note that our model assumed a hexagonal lattice, which limited the growth mechanism to hexagonal symmetry. This means that we cannot model topological defects such as rings of atoms with five or seven atoms. Additionally, our simulations began with an initial flake as a ribbon (measuring 0.284 × 100 nmgraphene . We did graphene ,92. Ther


In this photodetector, the Nb2O5 layer detects the light, and the graphene improves the responsivity based on the photogating effect. The photocurrent and the percentage ratio of the photocurrent to dark current of the Nb2O5 photogating photodetector are compared with those of the corresponding photoconductive photodetector. Also, the Nb2O5 photoconductive and photogating photodetectors are compared with titanium dioxide (TiO2) photoconductive and photogating photodetectors in terms of responsivity at different applied (drain-source) voltages and gate voltages. The results show that the Nb2O5 photodetectors have better figures of merit (FOMs) in comparison with the TiO2 ones.In the present study, by adding graphene to a photoconductive photodetector with a niobium pentoxide (Nb The first synthetic single-layer graphene was made and characterized by Game and Novoselov in 2004. For this invention, Game and Novoselov received the Nobel Prize in Physics in 20102. In 2013, two-dimensional atomic sheets of graphene and a new classification of nanoscale materials that can be used in electronics were proposed3. The exciting properties of graphene include high charge mobility, excellent thermal conductivity, and high strength7. One of the applications of graphene is in photodetectors. Single-layer graphene can absorb only 2.3% of the irradiated light from 300 to 2500 nm, so it has low responsivity8. The responsivity of graphene photodetectors can be improved in different ways, including the use of microcavity structures9, quantum dots10, graphene nano disc arrays11, graphene waveguides13, heterostructures, and graphene incorporated with different materials15. High responsivity was reported in the graphene photodetectors with photogating effect18. In 2010, a graphene detector was made. The graphene detector had a responsivity of 6.1 mA/W at a wavelength of 1.55 μm19. In 2012, a graphene detector with a cavity was presented. The detector had a responsivity of 21 mA/W at a wavelength of 850 nm9. In 2015, a graphene and boron nitrate detector with an optical waveguide was presented; this detector had a responsivity of 0.36 A/W20. Various studies have been conducted on graphene and light absorption processes, including photovoltaic effect (generating photocurrent based on electron–hole separation under the electric field at the junction of the regions with different impurities), photogating effect , bolometric effect (change in conductivity as a result of light radiation and temperature increase), and thermoelectric effect (creating voltage according to the Seebeck effect due to the increase in the temperature of carriers)21. Since the responsivity of the photogating effect is much higher than that of other effects, the present study focuses on this effect. In photogating effect, one of the carriers is trapped in the absorber layer. In other words, the lifetime of the additional carriers increases as the carriers are separated from each other by defects and impurities. If one type of the generated carriers is trapped, it can generate an additional electric field such as a gate voltage to modulate the channel conductance26. Such detectors with small dimensions show high responsivity and limited response speed due to the prolongation of the lifetime of the additional carriers27. In 2012, a graphene structure with quantum dots was proposed. In this structure, a responsivity of 107 A/W was obtained at a wavelength of 532 nm10. In 2016, graphene∕SiO2/lightly-doped Si structure was used for high sensitivity and responsivity. The operating range was from the visible to near-infrared regions, and the responsivity was 1000 A/W at a wavelength of 514 nm. In this photodetector, due to the defects between SiO2 and the lightly-doped Si, electrons accumulate in the traps and create a negative gate voltage, causing more holes to be induced and thus increasing a high gain25. In 2018, black phosphorus (BP) with a direct bandgap of 0.3 eV was used as a light-absorbing material. At wavelengths of 655 nm, 785 nm, and 980 nm, the responsivities of 55.75 A/W, 1.82 A/W, and 0.66 A/W were obtained, respectively. Excited electrons are trapped in trap levels, and the holes pass through the graphene layer by the internal potential between graphene and BP. The lifetime of carriers increases with traps. Due to the high mobility of graphene, holes can flow in the circuit before recombining with electrons. The introduced structure works in the visible to near-infrared regions based on the photogating effect22. In 2018, Ti2O3 nanoparticles with a band gap of 0.09 eV were used to fabricate a detector in the mid-infrared spectrum. The mechanism is the same as before. This detector had a responsivity of 300 A/W for a wavelength of 10 μm28. In 2018, the photogating effect in graphene photodetectors was investigated using SiO2/n-doped Si substrate. At wavelengths of 450 nm and 1064 nm, the responsivities were 500 A/W and 4 A/W, respectively. The bending of the band at the Si/SiO2 interface separates the electron–hole pairs. Under the electric field, the electrons move towards Si while the holes are trapped at the Si/SiO2 interface; the accumulation of holes at the Si/Si2 interface acts like a positive gate and increases the Fermi level of graphene. This causes the graphene to become n-type24. In 2018, a graphene transistor was fabricated with an indium antimonide (InSb) substrate. A responsivity of 33.8 A/W with a photogating effect at a wavelength of 4.6 μm was achieved29. In recent years various photodetectors with TiO2 and Nb2O5 absorber layers in the UVA region have been presented30. In 2011, an Nb2O5 nanobelt was proposed, and at 1 V, a responsivity of 15.2 A/W was obtained31. In 2015, an Nb2O5 nanoplate photodetector was fabricated with a responsivity of 24.7 A/W at 1 V32. In 2021, a MAPbI3 nanowire photodetector was fabricated, and a responsivity of 20.56 A/W at 1 V was reported33. In 2023, a type-II heterojunction of TiO2 NTs/Cs3Cu2I5 nanoparticles hybrid fiber was presented with a responsivity of 26.9 mA/W at − 1 V34. The photogating effect can be investigated in three different structures, namely quantum dot10, bulk25, and thin film structures8. Quantum dots can be integrated into two-dimensional materials to achieve some advantages. As the first advantage, quantum dots with greater thickness resolve the issue of the low optical absorption of two-dimensional materials. The second advantage is that two-dimensional materials have high carrier mobility, and the third advantage is that some two-dimensional materials do not have a wide absorption spectrum while quantum dots compensate for this limited responsivity. For a two-dimensional material such as graphene, there is no mechanism to produce multiple carriers from one photon. By using quantum dots, a large number of holes can flow in the circuit, and as a result, the gain increases. This is because the lifetime of the trapped electrons is long, and the mobility of carriers in graphene is high. One of the disadvantages of quantum dots is their toxicity. Moreover, the dimensions of quantum dots change the bandwidth of the used materials. In bulk detectors, due to the defects between SiO2 and lightly-doped Si, electrons accumulate in the traps and create a negative gate voltage, causing the induction of more holes and thus increasing the gain. In other words, the bending of the band at the Si/SiO2 interface separates the electron–hole pairs. Under the internal field, the electrons move towards the Si substrate while the holes are trapped at the Si/SiO2 interface, and the accumulation of holes at the Si/SiO2 interface acts like a positive gate and increases the Fermi level of graphene. As a result, the graphene is converted into n-type graphene. A highly-doped silicon substrate is not used since it has additional carriers with a much shorter lifetime. The application of the bulk structure is limited to high-energy materials and X-rays25.Two-dimensional materials can interact strongly with light. One of these two-dimensional materials is graphene, which is a monolayer of carbon atoms tightly arranged in a crystal network with a two-dimensional honeycomb pattern2O5 (3.7 eV) and TiO2 (3.2 eV) absorber thin films are used based on the photogating mechanism. The use of broadband materials is an advantage for the photodetector because it operates at room temperature. By transferring graphene to the photoconductive detector, the responsivity increases approximately 20 times. The use of an Nb2O5 absorber layer is a new technique. The advantages of this technique are its low cost and simplicity of obtaining Nb2O5 without any special technology only by combining a few solutions. In the present study, a photoconductive detector with an Nb2O5 absorber layer and a graphene photogating photodetector with the same Nb2O5 absorber layer are compared in terms of responsivity and the percentage ratio of photocurrent to dark current. The responsivities of the photogating and photoconductive detectors with the Nb2O5 absorber layer are 12.69 A/W and 0.65 A/W respectively. The percentage ratios of photocurrent to dark current of the photogating and photoconductive detectors with an Nb2O5 absorber layer are respectively 2.84% and 0.16% at a drain-source voltage of 1.5 V and a gate voltage of 1 V. The responsivities of the TiO2-based photoconductive and photogating detectors, which are fabricated under the same laboratory conditions, are 0.45 A/W and 8.32 A/W, respectively. The percentage ratios of the photocurrent to dark current of the TiO2 photoconductive and photogating detectors are 0.16% and 2.84%, respectively. The responsivity of a photogating detector with an Nb2O5 absorber layer is about two times higher than that of a TiO2 photogating detector.In the present study, Nb2O5 absorber layer are shown in Fig. 2. As shown in Fig. 2 layer by electron-beam physical vapor deposition. Then, as shown in Fig. 2O5 absorber layer on SiO2. For the anodization of Nb, an electrolyte consisting of 1200 ml of ethylene glycol C2H6O2, 800 ml of H2O, and 160 g of (NH4) B5O6 was used. The Nb layer was connected to the positive pole and the platinum electrode, which was inside the anodization solution, was connected to the negative pole, and the anodization process was carried out. As shown in Fig. Total is the total width of the transistor, n is the number of distances, and w is the width of the transistor.The fabrication steps of the photoconductive and photogating photodetectors with an Nb36.To fabricate the photogating photodetector, all the steps are the same as performed for the photoconductive photodetector; only at the end, as shown in Fig. 2O5 heterostructures. By placing the graphene monolayer on the Nb2O5 layer, due to the difference in the Fermi level of the two materials, a built-in potential barrier is created between the two materials. Then, light radiation in the range of the band gap of the Nb2O5 layer leads to electron–hole pair production inside the Nb2O5 layer. Due to the potential barrier between the graphene and Nb2O5 layer, the electrons move towards the graphene layer, but the holes are trapped inside the Nb2O5 layer. The trapping of the holes changes the Fermi energy level of graphene and the resistance of the graphene channel, causing a large photocurrent26.Figure N-Methyl-2-pyrrolidone (NMP) solution at a temperature of 70 Graphene was transferred through a wet transfer method in seven stages, including etching the copper layer, substrate preparation, and graphene transferring. The process begins by eliminating the unwanted graphene that is placed under the copper layer during the CVD process. This process was done in a 20% nitric acid solution in which the substrate was kept for about five minutes. Then, the copper was etched using 0.2 M iron (III) chloride for almost two hours. After the metal residues were removed by RCA, the substrate was ready for graphene transferring, but before that, the substrate must be prepared. Next, the substrate was immersed in piranha solution at 70 2 layer and interdigit electrodes to the Nb2O5 layer. Figure –6 Torr to remove the PMMA residues. The average graphene sheet resistance was obtained by the van der Pauw method. An HP4450 semiconductor measurement device was used at a voltage of 1 V and a current of 1 μFigure 27. The gain can be obtained using Eq. , the extra electron reaches the anode, and another electron immediately enters the photoconductor to maintain the charge neutrality and drifts to the anode terminal. This process is repeated until the extra electron recombines with a hole. This process takes τlife on average, and the gain is greater than unity. However, if τlife < τtr, the extra electron recombines with a hole before the transit is completed. To achieve a gain of greater than unity without multiple electron–hole pair production, a higher power by an external circuit is needed. Equation  in the present study. The oxide-to-metal conversion factor of Nb is 2.7, meaning that for 1 nm of Nb, 2.7 nm Nb2O5 is formed.GS is the control voltage.To increase the current, materials with a high absorption coefficient are used because according to Eq. , with in to Eqs.  and 8),,ox decreSome of the figures of merit (FOMs) of the photodetectors are evaluated optically. The optical characteristics, except for some special cases, were obtained using a helium-cadmium laser at a power of 1 μW, a wavelength of 325 nm, and a chopper frequency of 3 kHz. As shown in Fig. 2O5 and TiO2 absorber layers were optically examined.Using this optical setup has two advantages. The first advantage is that the ambient noise is eliminated, and the second advantage is that even the lowest amount of photocurrent can be observed with this setup. The photoconductive and photogating photodetectors with Nb2O5 layer was obtained by anodizing with an oxide-to-metal conversion factor of 2.7. The Ti layer was deposited on the silicon substrate by sputtering, and TiO2 was obtained by the oxidation method with an oxide-to-metal conversion factor of 1.7.As shown in Table 2O5 photoconductive detector, and Fig. 2O5 photogating detector. As shown in Fig. DS) is applied to both ends of the graphene layer, and the control voltage (VGS) is applied to the silicon substrate through ohmic contacts.Figure 2O5 absorber layer. The photocurrent of the photoconductive photodetector increases from 0.533 to 0.64 μA. By adding graphene, the photocurrent increases from 7.87 and 12.69 μA in the drain-source voltage range of 0.1–1.5 V. As the value of VDS increases, due to the reduction in the potential barrier, electrons more easily travel from Nb2O5 to graphene, and as a result, the increase in Iph is greater. In other words, according to Eq. , the carrier trapping time increases, and thus, the responsivity and photocurrent increase. The responsivity is also dependent on the initial energy level of the graphene layer. At a drain-source voltage of 1.5 V, the ratio of responsivity of the Nb2O5/Gr photodetector to that of the TiO2/Gr photodetector is about 2, and the ratio of the responsivity of the Nb2O5 photodetector to that of the TiO2 photodetector is about 1.5. This indicates the better performance of the Nb2O5 absorber layer compared to the TiO2 absorber layer while both of these layers were made under the same laboratory conditions.Figure 2O5 absorber layer are higher than those with the TiO2 absorber layer. The value of As shown in Table GS. However, in the photogating detectors, the carrier trapping time increases due to the photogating effect, so responsivity and photocurrent increase. The responsivity is also dependent on the initial energy level of the graphene layer. Therefore, it is proven that the increase in the responsivity of the photodetector with increasing VGS is because of transferring the graphene layer to the TiO2 absorber layer.Figure 2O5 and TiO2 photogating detectors are located at 20 V and 15 V, respectively. At the Dirac points, not only the photogating detectors have the highest responsivity, but also the curves have the highest slope around the Dirac points. As shown in the curves of the photogating detectors, further increase in the gate voltage has a downward trend, and responsivity decreases. The reason for this is shown in Fig. d/∂Vg, and the maximum of the responsivity curve, which is shown in red, is located at the points with the highest slope.As shown in Fig. 2O5 absorber layer was calculated as an FOM. With the increase in frequency, the frequency response criterion does not decrease substantially. These results indicate that both photodetectors can be used in applications, such as UV imaging and many modern systems, with a frequency requirement of up to 5 kHz.As shown in Fig. 2O5/TiO2 absorber layers (presented in this study) and previous detectors presented in other articles with the same absorber layer materials. According to this table, detectors with the Nb2O5 absorber layer have better responsivity than those with the TiO2 absorber layer. The Nb2O5 Nanobelts and the Nb2O5 nanoplates detectors have a better responsivity than the thin-film Nb2O5 photoconductive and photogating detectors. The Nb2O5 nanoplates detector has a better responsivity than the Nb2O5 Nanobelt, but the fabrication of the thin-film Nb2O5 detector is simpler than Nb2O5 nanoplates and nanobelts detectors.Table 2O5 absorber layer was used for fabricating a graphene detector based on the photogating effect. Moreover, an anodizing process was employed to produce this absorber layer from a thin layer of Nb. These processes have not been conducted in the literature and were presented in this study for the first time. It is worth noting that compared to photodetectors with a TiO2 absorber layer, the photodetectors with an Nb2O5 absorber layer performed better in terms of the cost, simplicity of the fabrication process, and high value of responsivity. With 325-nm laser radiation, 1 μW power, at a drain-source voltage of 1.5 V, and a gate voltage of 1 V, the responsivities of the photoconductive and photogating detectors with the TiO2 absorber layer were 0.45 A/W and 8.32 A/W, respectively, while the responsivities of the photoconductive and photogating detectors with the Nb2O5 absorber layer were 0.65 A/W and 12.69 A/W, respectively. Moreover, the percentage ratios of the photocurrent to dark current of the photoconductive and photogating detectors with the TiO2 absorber layer were 0.003% and 0.111%, respectively. However, the percentage ratios of the photocurrent to dark current of the photoconductive and photogating detectors with the TiO2 absorber layer were 0.16% and 2.84%, respectively. Therefore, the responsivities and the percentage ratios of the photocurrent to dark current in detectors with the Nb2O5 absorber layer were better than the detectors with the TiO2 absorber layer.An Nb


The historical insights and background of the discovery, development and clinical use of deferiprone (L1) and the maltol–iron complex, which were discovered over 40 years ago, highlight the difficulties, complexities and efforts in general orphan drug development programs originating from academic centers. Deferiprone is widely used for the removal of excess iron in the treatment of iron overload diseases, but also in many other diseases associated with iron toxicity, as well as the modulation of iron metabolism pathways. The maltol–iron complex is a recently approved drug used for increasing iron intake in the treatment of iron deficiency anemia, a condition affecting one-third to one-quarter of the world’s population. Detailed insights into different aspects of drug development associated with L1 and the maltol–iron complex are revealed, including theoretical concepts of invention; drug discovery; new chemical synthesis; in vitro, in vivo and clinical screening; toxicology; pharmacology; and the optimization of dose protocols. The prospects of the application of these two drugs in many other diseases are discussed under the light of competing drugs from other academic and commercial centers and also different regulatory authorities. The underlying scientific and other strategies, as well as the many limitations in the present global scene of pharmaceuticals, are also highlighted, with an emphasis on the priorities for orphan drug and emergency medicine development, including the roles of the academic scientific community, pharmaceutical companies and patient organizations. Iron is one of the essential metal ions found in all cells of the body, as well as in cancer cells and microbes. It is generally required for many enzymes and other proteins performing vital bodily functions and is involved in many physiological and biological processes, such as oxygen transport, storage and utilization; energy transduction; growth and development; lipid metabolism; and DNA synthesis ,2,3.Iron is tightly controlled in the human body using different mechanisms, pathways and proteins, which are involved in its uptake, distribution, utilization, recycling and excretion. Each cell requires and utilizes different amounts of iron and also other essential metal ions such as zinc and copper for different biological functions ,4,5,6,7.There is wide variation in the requirements for iron and other metals for different cell types, which in the case of iron is mainly reflected by the number of transferrin receptors on the cell membrane ,10,11,17It is estimated that about 4.5–5.0 g of iron is present in a 70–75 kg normal adult man, which is distributed in the blood and different organs. Most of the iron is in the form of heme in the proteins hemoglobin (2.3–2.6 g) in red blood cells (RBC) and myoglobin (0.32–0.40 g) in muscle. Iron is also distributed in the body, mainly in the form of polynuclear ferric oxyhydroxide phosphate complexes in the iron storage proteins ferritin (0.7 g) and hemosiderin (0.3 g), which are mainly found in the liver, spleen, muscles and bone marrow. Smaller amounts of iron are also found in mitochondrial cytochromes (17 mg), catalase (5 mg), transferrin (4 mg) and non-heme iron-containing enzymes (0.1 g) .2+) or ferric (Fe3+) state forms, and is always bound to different ligands containing oxygen, nitrogen and sulfur [−18 mol/L), since it mostly precipitates by forming insoluble polymeric ferric oxyhydroxide complexes with high stability constants (log K = 38) [At normal physiological conditions and pH levels, iron is mainly found in the ferrous (Fed sulfur . Under t K = 38) . In geneIron imbalance is associated with serious clinical conditions affecting many categories of patients ,3,20,21.There are many other diseases related to iron metabolism imbalances in addition to IDA, including the hemoglobinopathies and idiopathic hemochromatosis, which are the most common genetic diseases affecting humans ,47,48,49The major form of treatment of thalassemia major is regular red blood cell transfusions every 1–4 weeks and daily chelation therapy ,51. MultThere are many other diseases related to iron and other metal imbalance and toxicity conditions, where iron-chelating drugs and iron chelator complexes could be used as originally suggested in 2003 . Many ofDeferiprone and the tri-maltol–iron complex, also known as ferric maltol, Feraccru or Accrufer, were originally designed, synthesized and screened in vitro and in vivo in 1981 following the initiation of an academic project in 1978 ,61,62,63The path of development and the journey through the years of each of these drugs were completely different, with many difficulties and many intriguing turns of events related to research findings and the involvement of different academic centers, pharmaceutical companies, physicians, research investigators and patient organizations, which continue today ,63.The design and development of L1 and the maltol–iron complex were initiated in the biological chemistry department of the University of Essex, United Kingdom (UK), while working on a hemoglobin project in 1979 ,64. The Based on these prototype structures of alpha-ketohydroxypyrone and alpha-ketohydroxypyridine classes of chelators, about half a dozen new compounds were initially synthesized, including L1, and about a dozen commercially available compounds including maltol were screened in vitro in different models for their iron-binding and other properties . The in Due to lack of different screening facilities at Essex University for investigations related to the chelating compounds, including those for cell and in vivo studies, the developmental program for the iron chelators was partly moved to University College London Medical School (UCLMS) through contacts with the UKTS and associated researchers in UCLMS. In this context, new experimental screening programs were developed and showed that L1 was the most effective chelator for iron mobilization from transferrin and ferritin and also from iron-loaded mice following oral and intraperitoneal administration routes . SimilarThe situation was particularly urgent and frustrating regarding the development of orally administered L1 in thalassemia, since many patients’ lives were at risk because of complications with the 8–10 h long subcutaneous (sc) administration of DF, including compliance, toxicity and efficacy concerns . In the The pressure by thalassemia patients and parents was mounting for the rapid development of oral chelation therapy by the early 1980s. As a result, in 1985, the UKTS sponsored the continuation of the iron chelation project at the Royal Free Hospital School of Medicine (RFHSM) of the University of London from where the first publications of the iron removal effects of L1 in comparison to parenteral DF in mice and rabbits and later in other animal species were reported ,71,72,73The higher levels of safety and efficacy of L1 in different animal species in comparison to many other alpha-ketohydroxypyridines studied at the RFHSM, and several other developments such as the simple inexpensive synthesis of L1 and other alpha-ketohydroxypyridines, prompted the initiation of an application to the UK governmental regulatory authorities for clinical testing ,79. The In the meantime, several kilograms of L1 were prepared using the new method of synthesis, which was encapsulated in 0.5 g quantities by the pharmacy of Royal Free Hospital using gelatin capsules . The encIn the meantime, fierce competition against the development of L1 was initiated by academic and commercial groups, which began from the time of discovery and continues today for different reasons ,63. For Following the initial clinical trials at the RFHSM with L1, many other academic hospitals caring for thalassemia and other categories of transfusional iron-loaded patients worldwide expressed a great interest in joining the clinical trials and testing L1 in their institutions using the same protocol. In most cases, the vast majority of patients selected for the clinical studies were not responding or were intolerant to sc DF therapy. In many of the external clinical studies, L1 was supplied from the RFHSM  to instiWhile the clinical trials with L1 were continuing and expanding worldwide, an incidence of agranulocytosis was reported in 1989 in a Blackfan–Diamond patient at the RFHSM, causing the postponement of the L1 trials at the RFHSM to the dismay of the UKTS’ investigating team . SeveralDespite the encouraging pre-clinical and clinical results with L1, there was insufficient interest from major pharmaceutical companies for its commercial development, mainly because of financial considerations and especially the small number of thalassemia patients that require treatment in developed countries. In the meantime, many steps were taken to further advance the development of L1, including an application by the inventor proposing an international non-proprietary (INN) name for L1 in 1991, which resulted in the adoption by the World Health Organization (WHO) of the INN name deferiprone .Unexpectedly, the major breakthrough in the pharmaceutical development of L1 came from India, which played a leading role in the pharmaceutical development and distribution of the drug in developing countries. This development came about following a collaborative project that was initiated between a parent of a thalassemia patient and member of the Thalassemia Society in Mumbay, India, and the UKTS-sponsored group at the RFHSM. The parent also worked at the Indian generic pharmaceutical company Cipla, which was one of the leading generic drug manufacturers in India ,95,98. IAt about the same time as a BBC TV program highlighting the developments in India, the administration of the RFHSM decided to terminate all research on thalassemia supported by the UKTS, including the research on L1 and chelation therapy in general. This was a major drawback in the ongoing projects of L1, which continued elsewhere in the following years. Similarly, the BTG abandoned the development of all other chelating drugs except L1 and licensed the L1 invention to the generic pharmaceutical company Apotex, Canada, which received regulatory approval from the EU authorities for the use of L1 in 1999, followed by many other countries worldwide and also the USA FDA authorities in 2011, following the intervention of the USA Thalassemia Organization ,105,107.The academic and commercial conflicts regarding the use of L1 continued following the developmental stage and later introduction of DFRA in 2005 by Novartis, which was also the initial and exclusive manufacturer of DF for many years before the introduction of L1 ,110,111.The absence of full formal animal or other pre-clinical toxicology studies in the case of L1 by either Cipla or Apotex pharmaceutical companies has put L1 at a “theoretical disadvantage”, and for that reason it was initially classified as a “second-line” iron-chelating drug in comparison to DF and DFRA ,90,91,92With regards to its safety, long-term studies and continuous clinical monitoring involving thousands of thalassemia and other categories of patients for the last 30 years have confirmed the low toxicity levels of L1 ,120. TheDeferiprone’s ability as a monotherapy or in combination with DF to clear all excess iron and maintain normal iron stores using personalized dose protocols in regularly transfused thalassemia major patients signifies the complete treatment of iron overload and the achievement of the major aim of iron chelation therapy  119,120,122,123.Unlike the discovery and development of deferiprone, which was regarded as a life-saving drug for thousands of iron-loaded patients, especially in developing countries, the development of the maltol–iron complex was not urgently pursued by pharmaceutical companies. The delay was caused by commercial considerations and especially the availability of a large number of competing iron formulations used for the treatment of IDA, a condition generally considered non-life-threatening ,26.The discovery of the maltol–iron complex and its proposed use in IDA originated from the identification in 1979–1982 of the naturally occurring plant product and phytochelator maltol as an alpha-ketohydroxypyrone chelator, which was widely available and used in humans . The selFurther developmental studies involving the in vivo confirmation of the effects of the maltol–iron complex in relation to IDA were conducted following the original studies and design phase of 1979–1982 . These iIn a set of different experiments in rats, increased iron (59-Fe) uptake was observed following the intraduodenal administration of the iron (59-Fe) maltol in comparison to other hydrophilic iron (59-Fe) mixtures with fumarate, gluconate and EDTA . The oraDespite some initial preliminary interest about the development and commercialization of the maltol–iron complex for IDA by pharmaceutical companies, further animal and clinical studies, including long-term toxicology studies, were not pursued, probably because of the overall expenditure required for a new drug’s development at the time. No further efforts for the pharmaceutical development of the maltol–iron complex were undertaken, and the interest was focused on the pharmacological research aspects of maltol and its iron complex and other metal complexes  33,60,1,160,127.Many categories of patients have received the maltol–iron complex in a number of clinical studies, including for Crohn’s disease, ulcerative colitis, chronic kidney disease, pulmonary hypertension and also patients intolerant to other iron formulations ,138,139.There are several other advantages in the use of the maltol–iron complex over other iron formulations, including rapid iron absorption, low toxicity and higher selectivity in iron delivery in comparison to other iron formulations. These advantageous results, cause a decrease in the toxicity associated with the excess non-absorbable iron, which is usually observed with other iron formulations. In addition, the efficacy and low toxicity of the maltol–iron complex could benefit many patients experiencing complications and toxicity with other iron formulations, including intravenous iron and ferrous sulphate ,142,143.The level of safety of the maltol–iron complex appears in general to be satisfactory. This observation has also been reported in almost all of the clinical studies and trials, whereby the maltol–iron complex has been shown to be tolerable and effective, with significant improvements in anemia for the different categories of patients treated. In this context, the most common adverse effect during the clinical studies was mild gastrointestinal intolerance ,131.Overall, the maltol–iron complex may have many general advantages over other iron formulations for the treatment of IDA, which include the ease of administration, low toxicity, short timing for achieving the normalization of hemoglobin levels and low cost. In particular, the low cost of maltol–iron formulations and their general availability are major concerns affecting global health, considering that the vast majority of IDA patients live in developing countries with low health budgets. Furthermore, the low costs of maltol and iron may encourage the development of many different maltol–iron formulations, which could overturn any proprietary and other claims similar to generic pharmaceuticals ,145,146.While the major efforts in the development of L1 and the maltol–iron were based on the treatment of transfusional iron overload and IDA, respectively, their possible applications in other diseases were also suggested and tested at the early stages of their development, thereby contributing important information in the overall pharmacology and toxicology of both of these two drugs.The additional fields of investigation related to these two drugs were broadly based on other diseases of iron metabolism, infectious diseases, conditions related to free radical pathology and metal toxicity conditions other than for iron. In the meantime, many other analogues of L1 and iron–maltol were synthesized and tested for comparison with the original findings. The investigations included in vitro, in vivo and clinical studies.Thousands of patients suffering from iron overload and also other illnesses, as well as normal volunteers, have been treated in the last 40 years with L1, which is regarded as one of the safest drugs in the world per dose, e.g., 50–100 mg/kg received on a daily basis. In this context, the major category of patients receiving L1 is β-thalassemia major patients, followed by many other categories of iron-loaded patients, including β-thalassemia intermedia, HbE β-thalassemia, HbS β-thalassemia, sickle cell anemia, myelodysplastic syndrome, aplastic anemia, Fanconi’s anemia, Blackfan–Diamond anemia, pyruvate kinase deficiency, idiopathic hemochromatosis, iron overload in hemodialysis, and juvenile hemochromatosis ,153,154.Similarly, many other categories of patients with normal iron stores have also been receiving L1, mostly in short- but also in long-term clinical trials. The repurposing of L1 for diseases other than iron overload was organized based on similar dose protocols to those of iron overload and took place originally in the in the UK (RFHSM) but also other countries within a few years of initiating the clinical trials with transfusional iron-loaded thalassemia and myelodysplasia patients ,81,82,83In general, no serious toxicity was reported for clinical trials of up to 9 months duration and with a maximum dose of 100 mg/kg/day in patients with normal iron stores. Most importantly, significant clinical improvements have been reported in most of these categories of patients ,165,166.However, it is unfortunate that in many of the recent clinical trials involving mainly neurodegenerative disease patients, no rationale for the selection of the L1 dose protocol nor of iron metabolism balance studies was given ,159,160.It can be suggested in general that the positive results in terms of safety and the diverse categories of iron-loaded and other patients treated in clinical trials with L1 have increased the prospects of the wider use of the drug in many other diseases, including diseases related to free radical pathology and also in cancer ,172,173.The identification of the possible applications of maltol and the maltol–iron complex in medicine was proposed following their physicochemical characterization, protein and cellular interactions and in vivo and other studies . Within The maltol–gallium complex  has attracted a lot of interest because it has reached the stage of clinical trials and veterinary use, mainly for its anticancer and antimicrobial activities ,180,181.Another major area of application to medicine in relation to iron and other metal complexes of maltol is their use in the theranostic and diagnostic fields, which are rapidly expanding, involving many diseases ,191,192.The increased number of applications of maltol–metal complexes in medicine and veterinary medicine, as well as the pharmacological and toxicological information obtained from relevant studies, has increased the prospect of their wider use in many other fields of diagnostic and theranostic medicine ,198,199There are increasing prospects for the wider application of L1 and the maltol–iron complex in medicine, including for the optimization of existing therapeutic applications involving improved dose protocols, as well as combinations with other drugs or natural products, personalized medicine adjustments, the treatment of more and different clinical conditions and also more diagnostic and theranostic applications. In particular, there are many metabolic pathways and associated diseases that can further be investigated and targeted, with some involving iron interactions and free radical pathologies, many diseases related to metal metabolic pathways and toxicity other than iron, metal supplementation in essential metal ion deficiency diseases and new drug formulations and routes of administration. Many of these new applications have been suggested from relevant in vitro and in vivo studies and also following new clinical evidence ,173,186.The emphasis on new clinical applications of many drugs, including both L1 and the maltol–iron complex, is usually related to orphan diseases and diseases with no effective treatments . In thisExamples of previously established cancer targets include the classical restriction or reduction of the iron supply, inhibition of transferrin iron delivery and inhibition of ribonucleotide reductase in DNA synthesis, the high antioxidant potential by inhibiting free radical formation etc., ,206,207.In contrast to the mode of action of L1 in the above targets, lipophilic chelator metal complexes similar to maltol–iron and also other more lipophilic iron complexes have been proposed for the induction of ferroptosis in cancer cells, especially in cases of refractory or recurring tumors, and also in cases of drug resistance and metastasis  173,188188,204. The prospect of the application of L1 in infectious, inflammatory and many other diseases in addition to cancer has been previously reviewed ,183,184.Neisseria meningitides or cancer types such as glioblastoma, both of which depend on iron supply for growth [In general, the characteristics of the targets can impose many limitations in the selection of therapeutics, including chelating drugs for the optimization of treatments. For example, only L1 from all three iron-chelating drugs  can cross the blood–brain barrier and target infections in the brain, such as meningitis caused by r growth ,20,173.There are many other areas of potential development and applications of L1 and maltol–iron complexes. For example, intravenous L1 and maltol–iron complex formulations may have to be designed to benefit cancer and other categories of patients unable to receive oral formulations ,234,235.Despite the many difficulties and controversies, L1 and the maltol–iron complex are prime examples of academic drug discovery and orphan drug development medicinal products. In particular, L1 has helped in the treatment and survival of hundreds of thousands of iron-loaded and other patients worldwide in the past 30 years. Similar results are expected from the use of the recently approved maltol–iron complex for the treatment of IDA, which affects 1 in 3 or 4 individuals worldwide.The repurposing of L1 in many categories of non-iron-loaded patients, including neurodegenerative, infectious, inflammatory, metal toxicity and other conditions, signifies the safety and multitargeting potential of the drug. Similarly, the use of the maltol–gallium complex in cancer and also of other maltol–metal complexes in other therapeutic, theranostic and diagnostic applications highlights the future routes of development and use of new therapeutic, theranostic and diagnostic maltol and other chelator metal complexes.New advancements in the pathology of free radicals, cancers and many other diseases, including the discovery of new iron and other metal metabolic routes and proteins that can be modulated by L1, maltol–iron and also other new chelators and metal complexes, underlines the importance of chelators and chelator metal complexes in medicine. In particular, their use as monotherapies or in combinations with other drugs is essential for the improvement of the treatment of many diseases and increased prospects of patients’ survival, especially in many diseases with no effective therapies.


Abnormal deposition of extracellular matrix (ECM) in lung tissue is a characteristic of idiopathic pulmonary fibrosis (IPF). Increased collagen deposition is also accompanied by altered collagen organization. Collagen type XIV, a fibril-associated collagen, supports collagen fibril organization. Its status in IPF has not been described at the protein level yet. In this study, we utilized publicly available datasets for single-cell RNA-sequencing for characterizing collagen type XIV expression at the gene level. For protein level comparison, we applied immunohistochemical staining for collagen type XIV on lung tissue sections from IPF patients and compared it to lung tissue sections from never smoking and ex-smoking donors. Analyzing the relative amounts of collagen type XIV at the whole tissue level, as well as in parenchyma, airway wall and bronchial epithelium, we found consistently lower proportions of collagen type XIV in all lung tissue compartments across IPF samples. Our study suggests proportionally lower collagen type XIV in IPF lung tissues may have implications for the assembly of the ECM fibers potentially contributing to progression of fibrosis. Currently, the median survival time of patients with IPF is less than for many cancers, and there is no available cure1. As the lung ECM structure in IPF is drastically altered and provides a feedback loop that perpetuates disease progression3, investigating the underlying matrix pathology is expected to improve our understanding of the disease.Idiopathic pulmonary fibrosis (IPF) is the result of aberrant deposition of extracellular matrix (ECM) in the alveolar septa, causing restricted breathing in patients5. Among other types of collagens, collagen type XIV (COL14) is a fibril-associated collagen with interrupted triple-helices (FACIT)6 which consists of two collagenous domains (COL1 and COL2), which are important for the formation of the COL14 triple helical structures and for the binding to other fibrillar collagens, and three non-collagenous (NC) domains 7. Roles for COL14 have been suggested in regulation of fibrillogenesis, cell proliferation and differentiation9. The absence of COL14 in mice resulted in dysregulated biomechanical properties of the skin and altered fibril formation in the tendon6. Culture of human skin and mouse 3T3 fibroblasts on immobilized COL14 showed that COL14 inhibits cell proliferation and triggers the differentiation of 3T3 fibroblasts8. The presence of COL14 has been demonstrated previously in several tissues including skin, tendon, cornea and lung; especially in the vicinity of the blood vessels, airway smooth muscle cells (ASM) and bronchial epithelium10. In previous studies by Nishiyama et al.11 and Gordon et al.12, a role for COL14 in the regulation of mechanical stress has been suggested. We have previously reported the localization of COL14 in regions that would be subjected to high mechanical stress in the lung including the parenchymal region, blood vessels, ASM and bronchial epithelium10. Therefore, the presence or absence of COL14 could impact the fibrillogenesis and organization of the collagen type I fibrils and the biomechanical properties of the ECM in different lung compartments and subsequently the lung function.Deposition of collagens in the altered ECM in IPF has been well documented, including an increase in collagen types I and III, as well as changes in the organization of collagen fibrils13. However, the time for development of fibrosis in this model compared to IPF and/or species differences can influence how these results translate to humans. Accordingly, in the current study we aimed to investigate whether there are differences in the COL14 gene expression and protein levels in lung tissue from patients with (end stage) IPF compared to normal human lung tissue.Unlike abnormal collagen (type I) deposition and organization in IPF which has been described in detail, the role of FACITs in this process or, more specifically, involvement of COL14 in IPF, remains unknown. Previously, in a mouse model of fibrosis, higher levels of COL14 deposition were shown in bleomycin-treated mice compared to control miceCOL14A1 was analyzed in lung tissue of patients with IPF and non-IPF donors using publicly available single-cell RNA sequencing datasets from the Human Lung Cell Atlas (HCLA) consortium and the IPF atlas dataset from Adams et al.15. These datasets were analyzed and clustered using HCLA consensus cell-type annotations for 4 major cell groups: endothelial, epithelial, immune and stromal cells for analysis of the expression profile of the Collagen Type XIV Alpha 1 Chain (COL14A1) gene which encodes COL14 protein.Cell-type specific gene expression of 10.IHC was used to quantify proteins of interest in lung tissue. Control lung tissues were obtained through resections from patients undergoing surgery for lung cancer, taken from as far away from the tumor region as possible and were macroscopically normal tissue as assessed by pathologists. Fibrotic lungs were obtained from explanted material following transplantations Table . Formalihttps://umcgresearch.org/w/research-code-umcg) and national ethical and professional guidelines ). Lung tissues used in this study were derived from leftover lung material after lung surgery and transplant procedures. These materials were not subject to the Medical Research Human Subjects Act in the Netherlands as confirmed by a statement of the Central Ethics Review Board of the University Medical Center Groningen; all biological samples are from archival materials that are exempt from consent in compliance with applicable laws and regulations  art 458/GDPR art 9/UAVG art 24). All samples and clinical information were coded before experiments were performed, blinding any identifiable information to the investigators.The study protocol was consistent with the Research Code of the University Medical Center Groningen  in the whole tissue of non-IPF and IPF lung samples using the same image analysis pipeline Fig. . CompariCollectively, our results show proportionally lower COL14 protein expression relative to other ECM components that make up lung tissue in patients with IPF compared to both ex-smoker and never-smoker non-IPF controls. To the best of our knowledge, this is the first report of protein level changes related to spatial location of COL14 in human lung tissue in IPF. In addition, we found a lower ratio of COL14 to COL1 in IPF lungs compared to never smoker non-IPF controls.16 suggests that such an absolute increase is likely. However, the ratio of COL14 to COL1 in the same study was lower in IPF lungs compared to control lungs, which is consistent with our findings. While an early study analyzing the amount of COL14 in IPF lungs semi-quantitatively reported higher COL14 in established fibrotic areas and lower COL14 in fibroblastic foci17, these results portray an absolute amount of protein rather than relative to other ECM components as depicted in our study.In this study, we examined the proportion of lung tissue positive for COL14 in each sample and not the absolute amount of protein. As the total amount of tissue present in IPF lung sections is larger than in controls, it is conceivable there is an increase in absolute amount of COL14, while the proportion of COL14 in comparison to other ECM components is reduced. A previous report of higher mass spectrometry counts of COL14 in IPF lungs compared to non-IPF lungs9 to investigate the changes in COL14A1 expression in lung tissue from patients with IPF compared to non-IPF controls have revealed a higher expression in fibroblasts and myofibroblasts, as well as in basal cells isolated from IPF lungs. These differences in COL14A1 mRNA expression between patients with IPF and controls are in the opposite direction than the differences observed at the protein level in our IHC stainings. It has been shown previously that correlations between the steady-state number of protein molecules per cell and their associated mRNA molecules are weak18. Proportional changes between the relative abundances of the proteins and the relative abundances of the mRNA of these proteins is also not guaranteed19, and possible mechanisms for this lack of proportion between mRNA and protein levels have been discussed elsewhere20. In addition, the status of the proteins already deposited in the ECM is unlikely to be determined by their mRNA. It might be possible that the increased gene expression of COL14A1 reflects an attempt of the structural cells of the lung to compensate for the reduced COL14 protein levels observed in lung tissue.The single-cell analyses using the IPF atlas21. Even with this, recent proteomics data from Hoffman et al. show a lack of absolute increase in COL1A1 in decellularized IPF lung samples22. An interesting observation from our study was the influence of smoking on the lung ECM proteins in the lungs of non-IPF donors. While relative amounts of COL14 did not differ based on the smoking status of the non-IPF controls, the percentage positively stained areas for COL1 differed significantly between the never smoker and ex-smoker groups. Considering that the lungs of ex-smoker donors did not have visible fibrotic regions, it is possible that this higher relative amount of COL1 reflects more absolute amounts. In IPF lung tissue, the samples had COL14 positive areas in dense fibrotic tissue while the fibroblastic foci and honeycomb regions were mostly devoid of COL14. Further research is needed to investigate how the collagen organization differs in these dense fibrotic tissue areas with respect to the COL14 presence as well as relating these changes to lack of COL14 in fibroblastic foci or honeycomb regions.Regarding our results from the COL1 image analysis, we did not observe a relative higher amount of the percentage positively stained areas in IPF lung samples compared to control groups. This lack of increase, however, does not mean a lack of absolute increase as more deposition of COL1 in IPF lungs has been previously established5. More research correlating fibrillar collagen organization with FACITs such as COL14 will increase understanding of the involvement of COL14 in the development and/or progression of fibrotic responses. A recent study using mice deficient in COL14A1 found increased stiffness and abnormal collagen fibril organization in the corneas of COL14A1-knockout mice compared to wildtypes23. These observations parallel well-documented increases in the stiffness of lungs from patients with IPF16. Together with our observations reported here, it is tempting to speculate that COL14 protein levels in IPF lungs do not mirror the increase in other ECM proteins in lung tissue during fibrosis, thereby leading to more disorganized collagen fibers with higher stiffness in lungs of patients with IPF.How COL14 is involved in the regulation of human lung ECM structure and how changes in its proportional expression influence fibrotic responses in IPF is currently unknown. As FACITs regulate the structural organization of fibrillar collagens, it can be argued that lower proportions of COL14 in IPF lungs contribute to the reported imbalance between organized and disorganized fibrillar collagen in IPFSupplementary Information.