Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease of the lung with poor survival. The incidence and mortality of IPF are rising, but treatment remains limited. Currently, two drugs can slow the scarring process but often at the expense of intolerable side effects, and without substantially changing overall survival. A better understanding of mechanisms underlying IPF is likely to lead to improved therapies. The current paradigm proposes that repetitive alveolar epithelial injury from noxious stimuli in a genetically primed individual is followed by abnormal wound healing, including aberrant activity of extracellular matrix–secreting cells, with resultant tissue fibrosis and parenchymal damage. However, this may underplay the importance of the vascular contribution to fibrogenesis. The lungs receive 100% of the cardiac output, and vascular abnormalities in IPF include (a) heterogeneous vessel formation throughout fibrotic lung, including the development of abnormal dilated vessels and anastomoses; (b) abnormal spatially distributed populations of endothelial cells (ECs); (c) dysregulation of endothelial protective pathways such as prostacyclin signaling; and (d) an increased frequency of common vascular and metabolic comorbidities. Here, we propose that vascular and EC abnormalities are both causal and consequential in the pathobiology of IPF and that fuller evaluation of dysregulated pathways may lead to effective therapies and a cure for this devastating disease. Idiopathic pulmonary fibrosis (IPF)  is a proThe pathogenesis of IPF is considered to result from injury to the alveolar epithelium by a range of insults including cigarette smoke, gastric acid, air pollution, and viruses in a genetically primed individual, driving aberrant activity of mesenchymal cell populations . SubsequVascular abnormalities in IPF include the development of vascular connections known as pulmonary-bronchial artery anastomoses and are accompanied by abnormal populations of endothelial cells (ECs) and vascular comorbidities such as pulmonary hypertension (PH) and coronary artery disease  Figure . HoweverMargaret Turner-Warwick described the expansion of the vasculature with numerous pulmonary-bronchial arterial anastomoses in diffuse pulmonary fibrosis as early as 1963  Figure . These aIt is, therefore, unclear whether the VEGF-inhibiting effects of nintedanib are undermining or augmenting its antifibrotic effects. In advanced disease states, the overall effect is a net reduction in pulmonary vascular surface area, which, in part, explains the increased frequency of PH. This loss of pulmonary vascular surface area is associated with distinct cellular changes in ECs, vascular smooth muscle cells (VSMCs), and pericytes, all of which may directly contribute to the pathogenesis of IPF .Alveolar injury is likely to directly injure ECs . ECs shaIn IPF, an abnormal, ectopic population of COL151A-expressing ECs have been identified using single-cell RNA sequencing . These CNumerous animal models have shed light on the role of the endothelium in pulmonary fibrosis. In one mouse model, pulmonary capillary ECs have been shown to support epithelial cell proliferation and alveolar regeneration through the secretion of factors including MMP-14, a process that is dependent on the activation of VEGF receptors present on ECs . BleomycECs may also be directly involved in fibrogenesis through endothelial-mesenchymal transition (EndMT) whereby they develop a contractile, α-smooth muscle actin–expressing phenotype . This efThese data suggest that following lung injury, damaged ECs secrete profibrotic mediators and are capable of migrating to the parenchymal regions of lung and transdifferentiating into pathogenic myofibroblasts. This model implies that ECs play a fundamental role in pathogenesis, serving as much more than mere bystander cells.VSMCs are highly contractile cells that constitute the structural form of blood vessels. There is abnormal proliferation and distribution of smooth muscle cells in IPF tissue , and undVSMCs isolated from patients with IPF show a hyperproliferative state and produce more collagen I via induction of reactive oxygen species compared with cells from control donors. This effect can be blunted by the antifibrotic therapeutic pirfenidone . The proAdenoviral overexpression of TGF-β1 in the lungs of mice leads to signaling crosstalk between ECs, VSMCs, and fibroblasts, which not only results in increased rates of apoptosis of ECs but also induces activation and proliferation of VSMCs. Importantly, defective bone morphogenetic protein receptor 2 (BMPR2) signaling has been implicated in VSMC activation and proliferation . In partPericytes are mesenchymal cells, closely related to fibroblasts, that form extensive physical contacts with ECs  with which they share a basement membrane . PericytThe alveolar epithelial-capillary basement membrane (BM) is a vital scaffold structure that supports normal repair of the alveolar parenchyma, and loss of BM integrity has been known for many years to be a fundamental pathogenic change in IPF . Loss of1 and α2 collagen IV chains. This change in BM composition limits myofibroblast migration and promotes their survival, which could contribute to myofibroblast persistence and sustained activity in fibroblastic foci  is expressed in vascular ECs and produces NO via a calcium/calmodulin pathway. NO subsequently activates soluble guanylyl cyclase (sGC) to generate cGMP, which in turn activates protein kinase G (PKG), leading to modulation/reduction of intracellular calcium concentration, leading to EC permeability, smooth muscle relaxation, and the inhibition of platelet aggregation . When eNThe sGC stimulator riociguat targets the NO/cGMP pathway and is licensed for use specifically in chronic thromboembolic pulmonary hypertension and also has demonstrable antifibrotic effects in animal models , 66. In Inhaled NO gas has recently been shown to improve exercise capacity (as assessed by ability to undertake moderate to vigorous activities) and was well tolerated in patients with ILD and PH (ILD-PH) where IPF represented the largest subgroup of ILD in a phase IIb RCT . HoweverEndothelin (ET) is a potent vasoconstrictor with vascular remodeling properties; therefore, ET antagonists are used in the treatment of idiopathic PAH. ETA and ETB receptors are expressed on alveolar epithelial cells and fibroblasts, both of which, under certain disease conditions, are capable themselves of ET synthesis . ET can The dual ETA and ETB receptor antagonist bosentan was compared with placebo in 158 patients with IPF in the BUILD-1 study. Bosentan was well tolerated; however, the trial did not meet its primary endpoint of improvement in 6-minute walk distance (6MWD) . HowevercAMP is an intracellular messenger formed by adenylate cyclase. In the endothelium, cAMP functions to maintain barrier junction integrity and permeability (through a combined effect with Rho) and vascular smooth muscle tone . In humaThe nonselective PDE4 inhibitor roflumilast is currently in use in chronic obstructive pulmonary disease and has proven effective in bleomycin mouse models in limiting fibrosis and vascular remodeling . HowevercGMP is a parallel intracellular second messenger to cAMP with similar functional effects in some systems. cGMP is catabolized by a number of PDE enzymes, with PDE5 being the most therapeutically valuable and a standard target in the treatment of PAH. Inhibition of PDE5 in in vitro models with sildenafil prevents TGF-β–induced EndoMT and VSMC-mesenchymal transition through downstream signaling on ERK1/2 and SMAD, providing further evidence of a beneficial antifibrotic effect of these pathways .In a small open-label study, beneficial effects of sildenafil on 6MWD observed in patients with IPF-PH suggested that enhancing NO/cGMP pathways has therapeutic potential in this condition . Subsequ2 (PGE2), PGI2 (prostacyclin), and PGD2, all formed from arachidonic acid (2 and converted to PGH2 by the action of cyclooxygenase 1 (COX-1) (constitutive) or COX-2 (inducible). PGH2 is converted to its respective prostanoid by the action of the site-specific synthase. In the vasculature, prostacyclin is the primary prostanoid produced, largely because of coexpression of COX-1 and prostacyclin synthase by ECs  (Prostanoids have long been investigated as possible antifibrotic mediators. Reduced levels of PGEwith IPF . In genewith IPF . This efF (CTGF) . cAMP alF (CTGF) . This syF (CTGF) . In VSMCF (CTGF) . In addiF (CTGF) .2 is a central component of the inflammatory response in humans. While a multitude of cells release the wider range of prostanoids, PGE2 is considered a critical regulator of inflammation, and inhibition of PGE2 at the site of inflammation explains much of the therapeutic benefit of NSAIDs. Animal models and in vitro experiments on human lung fibroblasts have demonstrated reduced production of COX-2–dependent PGE2, which may be explained by epigenetic changes in patients with IPF and the milieu of chemokines such as CCL2, which inhibits PGE2 release  expression in fibrotic tissue. PGE2 can inhibit fibroblast proliferation and ECM production through the EP2 and EP4 receptors in a cAMP/PKA-dependent manner; however, higher PGE2 concentrations can have a profibrotic effect via the EP1 receptor (through downregulation of cAMP) and the EP3 receptor . PGE2 can also inhibit the effect of TGF-β and the SMAD pathway. Administration of exogenous PGE2 in mice has a protective effect against bleomycin-induced fibrosis , leading to phosphorylation of myosin light chains to reorganize the actin cytoskeleton, promoting cell contraction, motility, and adhesion in inflammatory cells, smooth muscle cells (notably VSMCs that are important for regulating vascular tone), and platelets , 113. TwA mechanistic link has been demonstrated in shared pathways in RhoA activation and PDE4 via A-kinase anchoring protein 13 (AKAP13) . AKAP13 In animal models of fibrosis, inhibition of ROCK using fasudil  and the experimental compound Y-27632 attenuates fibrosis and vascular remodeling , 121. InThere are a number of potential mechanisms through which fibrosis and abnormal clotting may occur. Tissue factor initiates the extrinsic coagulation pathway, is highly expressed by alveolar epithelial cells in patients with IPF, and generates lung-tissue fibrin deposits, which serve as a platform for inflammatory cells and profibrotic cytokines, enhancing their accumulation at sites of injury . This envβ6 integrin  with placebo, demonstrated harm, and this detriment was due to accelerated fibrosis rather than bleeding complications . Recent ne model .vβ1 and αvβ6, which are implicated in PF and have important effects on EC function (Other EC-relevant pathways that have been investigated in PF include (a) autotaxin, the enzyme responsible for generation of the profibrotic lipid mediator LPA, which also has phosphodiesterase activity and is highly expressed by ECs; (b) the integrins, including αfunction ; (d) CTGfunction . Specififunction .It is evident from cell and molecular biology that the vasculature, and related signaling pathways, have an important role in the development of IPF. If the vasculature is playing a prominent role in the pathogenesis of IPF, one would hypothesize that there would be substantial systemic disease associated with IPF. While PH is a well-recognized complication of IPF, other comorbidities are gaining prominence. Indeed, systemic hypertension and diabetes are common comorbidities in IPF , while cThe epithelium — the primary site of initial insult in IPF — sits in close proximity to the endothelial layer in the alveolus, separated only by a thin basement membrane, which itself is abnormal in the condition. Injury to the former will undeniably affect the latter. There is compelling evidence from various in vivo, in vitro, and ex vivo models that aberrant endothelial responses occur with resultant loss of integrity of the basement membrane, vascular remodeling, and the generation of vascular signals further driving ECM deposition, fibrosis, and parenchymal lung damage. Whether it is the epithelial signal to the endothelium that initiates this process in its entirety, or whether the endothelium is the primary culprit, remains an area for development in the field of fibrosis research. However, given the findings, one cannot ignore the potential role of the circulation in the pathobiology of IPF.The numerous disrupted vascular-relevant signaling pathways present in IPF are now being explored in greater detail and mapped more clearly. The specific targeting of these pathways — especially with prostanoid agents and phosphodiesterase inhibitors — represents an expanding chapter in the treatment of this challenging disease. As closer associations between IPF and numerous more common cardiovascular and metabolic conditions are made, a deeper understanding of the disease and how to treat it is likely to ensue. Therefore, strategies that target endothelial repair by focusing on reprogramming abnormal metabolic responses may ultimately provide an opportunity to prevent the progression of, or potentially even reverse, the fibrotic response.


Cryptococcus neoformans and Cryptococcus gattii are the causative agents of cryptococcosis. Virulence and susceptibility to antifungals may vary within each species according to the fungal genotype. Therefore, specific and easily accessible molecular markers are required to distinguish cryptic species and/or genotypes. Group I introns are potential markers for this purpose because they are polymorphic concerning their presence and sequence. Therefore, in this study, we evaluated the presence of group I introns in the mitochondrial genes cob and cox1 in different Cryptococcus isolates. Additionally, the origin, distribution, and evolution of these introns were investigated by phylogenetic analyses, including previously sequenced introns for the mtLSU gene. Approximately 80.5% of the 36 sequenced introns presented homing endonucleases, and phylogenetic analyses revealed that introns occupying the same insertion site form monophyletic clades. This suggests that they likely share a common ancestor that invaded the site prior to species divergence. There was only one case of heterologous invasion, probably through horizontal transfer to C. decagattii (VGIV genotype) from another fungal species. Our results showed that the C. neoformans complex has fewer introns compared to C. gattii. Additionally, there is significant polymorphism in the presence and size of these elements, both among and within genotypes. As a result, it is impossible to differentiate the cryptic species using a single intron. However, it was possible to differentiate among genotypes within each species complex, by combining PCRs of mtLSU and cox1 introns, for C. neoformans species, and mtLSU and cob introns for C. gattii species.The species complexes  Cryptococcus neoformans and Cryptococcus gattii, which in nature can be found in different environments, such as bird excrement , soil, rivers, decomposing vegetation, and tree hollows [Cryptococcosis is a systemic/opportunistic mycosis caused by pathogenic basidiomycete yeasts from the species complexes  hollows ,2. The m hollows .Cryptococcus species, the necessity of more precise techniques for the diagnosis has led to the development of some molecular approaches, such as AFLP and PCR-RFLP of the URA5 gene, to encompass all genetic variability of clinical and environmental isolates [C. neoformans , C. deneoformans (VNIV), C. gattii (VGI), C. deuterogattii (VGII), C. bacillosporus (VGIII), C. tetragattii (VGIV), and C. decagattii (VGVI). The VNIII genotype is believed to be a hybrid between C. neoformans and C. deneoformans. Recently, Farrer et al. [C. gattii in forests of Zambia, in association with nitrogen-rich excreta from a small mammal, known as Hyrax (Dendrohyrax arboreus). This new genotype was named VGV.Regarding isolates ,5. The gisolates : C. neofr et al.  reportedC. neoformans and C. gattii isolates is of utmost importance, as there are variations in the susceptibility to antifungals among different genotypes. Clinical data suggest that the response to antifungal therapy is less responsive in C. gattii infections, requiring prolonged treatment, especially because they are associated with cryptococcomas formation [C. deuterogattii) is the least susceptible to antifungals , followed by VGI, VNI, and VNIV isolates [From a clinical perspective, accurately identifying the species or genotype of ormation ,9,10. Adisolates ,12,13,14C. neoformans and C. gattii complexes, the quest for novel and readily accessible molecular markers for genotyping becomes highly essential, and group I introns appear to be well-suited for such investigations, in view of the fact that they are polymorphic in their presence and sequence [Moreover, considering the cryptic speciation in the sequence . Those esequence , but somsequence . These Hsequence . Besidessequence ,21,22,23sequence .mtLSU gene, which encodes the large-subunit rRNA of the mitochondrial ribosome [C. neoformans and C. gattii, and to assess whether these introns possess informative polymorphism for distinguishing among genotypes or species complexes, we investigated the presence, polymorphism, and phylogenetic relationships of group I introns in the mitochondrial genes cox1 (cytochrome c oxidase) and cob (cytochrome b). These genes were selected because the current literature has demonstrated them to be the most frequently invaded by introns in fungal mitogenomes [In a recent study, our research team examined the presence of group I introns in the ribosome . We founogenomes .Cryptococcus isolates used in this study had already been genotyped by Gomes et al. [URA5 gene, as described by Trilles et al. [Cryptococcus neoformans  and 25 are Cryptococcus gattii . Two recent strains (HGT17 and HGT18) were isolated from patients hospitalized at Hospital Giselda Trigueiro in Natal, under the approval of the Research Ethics Committee of the Federal University of Rio Grande do Norte: CAAE 39640614.8.0000.5537, and they were genotyped in this study as mentioned above. The list of all isolates, their genotypes, and sources is available in Seventy-four of the seventy-six s et al.  by PCR-Rs et al. . Among tcob and cox1 genes were performed using manually designed primers , according to the manufacturer’s instructions. The thermocycling consisted of an initial cycle of 30 s at 98 °C for initial denaturation, followed by 35 cycles of 10 s of denaturation at 98 °C, 30 s of annealing at 53 °C, 1 min of extension at 72 °C, and a final extension cycle of 10 min at 72 °C. Electrophoresis was performed in 1% agarose gel  with ethidium bromide and visualized under a UV transilluminator . To confirm the presence of introns and endonuclease homing genes, some PCR products of different sizes, in the different genotypes, were selected and purified using the IlustraTM GFXTM PCR DNA and Gel Band Purification kit , according to the manufacturer’s instructions, and subjected to capillary electrophoresis sequencing at MACROGEN/South Korea, in order to confirm the presence or absence of the intron.https://megasun.bch.umontreal.ca/RNAweasel/), accessed on 20 January 2023, was used to classify the sequenced introns, according to their RNA secondary structure. The algorithm ERPIN [https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), accessed on 25 January 2023.As group I introns are divided into five classes, based on structural characteristics (IA-IE), the online tool RNAweasel   or the Mann–Whitney test (intron treated as quantitative data: number of introns). The statistical analyses were performed using Stata software (v. 11.1) and the significance level was established at cob gene are listed at the C. neoformans and C. gattii complexes, whose PCR products ranged from 600 to 1200 bp. Regarding the intron in the second insertion site of the cob gene, its presence was confirmed by the amplification of a 1500 bp band for majority of the isolates from the different genotypes, with the exception of the VNII and VGIV isolates studied here, which presented a 500 bp band, which refers to the flanking exons. The two bands, 500 and 1500 bp, for the absence and presence, respectively, of the second intron from the cob gene, were amplified for one VGIII and one VNI isolate, indicating a possible heteroplasmy, i.e., the presence of different mitochondria in the same cell.According to PCR-RFLPs of the URA5 gene, the two new isolates from clinical samples (HGT 17 and HGT18) belong to the VNI genotype. The size of PCR products for group I introns from the cox1 gene for each fungal isolate are listed at the cox1 gene, the 600 bp product (indicating its absence) was obtained for all VNI isolates. For the other genotypes, most of the isolates presented a 1500 bp band, indicating the presence of the intron. Some isolates  presented both bands, 600 bp and 1500 bp. The second intron was absent in almost all VNI and VGII isolates (200 bp band) and present in most of the isolates from the other genotypes (1200 bp band). Some isolates  presented both bands: 200 bp and 1200 bp. The third intron was absent in almost all VNI isolates and in all VNII and VGIV isolates (160 bp band). PCR products indicating the presence of the third intron presented 1200 bp for some VNI, VNIII, VNIV, and VGII isolates, 400 bp for a VGIII isolate, and 500 bp for a VGI isolate. Some isolates (1 VNIV and 15 VGII) also presented two bands (160 and 1200 bp). Regarding introns 4 and 5, they were amplified together, in one single PCR reaction. For this PCR, the band of 300 bp, which refers to the flanking exons only, was observed for all VNI and VNII isolates and for a few of the isolates belonging to the other genotypes. Bands indicating intron presence had different sizes: >1600 bp for the presence of both introns 4 and 5, 1400–1500 bp for the presence of intron 4 only, and 500 bp indicating the presence of intron 5 only. Signs of heteroplasmy were also detected here, including isolates with the three possible bands. The presence of more than one band was observed for 1 VNIII, all VNIV, for 17 of the 19 VGII, and for 1 VGIII  (https://blast.ncbi.nlm.nih.gov/Blast.cgi), accessed on 10 March 2023, which revealed that this intron shares significant similarities with introns found in other fungi. For example, it exhibited 78% identity, with a coverage of 20% and an e-value of 2 × 10−16, when compared to the fourth intron in the cox1 gene from Arthonia susa, a lichenized ascomycete. Furthermore, it demonstrated 74.73% similarity, with an 18% coverage and an e-value of 4 × 10−19, when compared to the fourth cox1 intron from Phellinotus piptadeniae, a wood-decay basidiomycete. Additionally, it showed 74.48% similarity, with a coverage of 31% and an e-value of 5 × 10−18, when compared to a nuclear sequence on chromosome 7 from Apiotrichum mycotoxinovorans (Trichosporon mycotoxinovorans), an opportunistic basidiomycete pathogen.The sequencing of some of the PCR products was conducted to compare their sequences to those used as references for primer design. The GenBank accession numbers for all the 36 sequenced introns are OQ946649 to OQ946684. One sequenced intron, from the FC9 isolate (VGIV), amplified with the primers flanking the second intron from For sequences containing introns, we observed size and sequence polymorphisms that were associated with the presence of open reading frames (ORFs) containing homing endonuclease genes (HEGs), all from the LAGLIDADG family. These ORFs were detected in 29 of the 36 sequenced introns.cox1 gene is only invaded by introns from the families IA and IB, while the cob gene only has introns from the families IA and ID.The primary sequence of each sequenced intron , as shown in The search for group I introns in the in mtLSU  on the smtLSU, two in cob, and five in cox1) was VNIII, a hybrid between the species C. neoformans and C. deneofomans (VNIV) [C. neoformans and the most commonly isolated genotype, from clinical specimens, in the world [s (VNIV) , while the world .C. neoformans and C. gattii complexes, there was no statistical significance between the amount of introns in the mtLSU gene, as previously mentioned [cob and cox1 genes, mainly for the intron 1 of cob, and the introns 1, 3, 4, and 5 of cox1  has been proven to be an outlier, considering its lower similarity to the other introns  and detected variations in size ranging from 24,740 to 31,327 bp, primarily due to differences in the number and size of mitochondrial introns, as well as the presence of HEGs [The PCR results confirmed that the presence or absence of self-splicing introns in the mitochondrial genes of  of HEGs .C. neoformans complex had fewer group I introns than C. gattii (p < 0.001) in the cox1 and cob genes. This represents a distinct pattern from what has been previously observed for the introns in the mtLSU gene [mtLSU gene, although it had a high frequency of introns in the cox1 and cob genes. The association between intron presence and virulence requires further investigation for introns in different mitochondrial genes through an experimental approach, using animal models.Another noteworthy finding is that, on average, the LSU gene . For insC. gattii is the difference in the mitochondrial inheritance mechanism between these complexes. For C. neoformans, it is mostly uniparental, while in C. gattii it is frequently biparental [One possible explanation for the high frequency of introns in parental . This coC. gattii may also be associated with the increased occurrence of multiple bands observed on PCR electrophoresis for a specific intron, a phenomenon that was more commonly observed in this species complex  has already been experimentally demonstrated by knocking out the transcription factor oformans . In typin et al.  observedtogenome ,42,43.cob and cox1 genes, can be explained by the presence or absence of group I introns, as well as the presence of homing endonuclease genes (HEGs) within these elements. HEGs were identified in 29 out of the 36 sequenced introns, all belonging to the LAGLIDADG family, according to CDD-NCBI. Once functional, these endonucleases contribute to the dispersion of their introns, leading to a super-Mendelian inheritance pattern in heterozygous crosses or, in some cases, they may also function as maturases, enzymes that stabilize the secondary structure of introns, assisting their auto-splicing [The size polymorphism of the intronic sequences observed in splicing ,45,46. Acob gene, and the IA and IB families for the cox1 gene. Notably, group I introns occupying the same insertion site from different fungal isolates were consistently identified as belonging to the same family, suggesting a single origin for all of them, possibly through ancestral invasion. To investigate this, individual phylogenetic analyses were conducted for the introns in the cob and cox1 genes . A BLAST search revealed that this intron is similar to introns from other fungi, such as to the fourth intron of the cox1 gene from Arthonia susa, an ascomycete-lichen, and from Phellinotus piptadeniae, a basidiomycete wood-decay fungi, and also to a nuclear sequence from Apiotrichum mycotoxinovorans, an opportunistic basidiomycete yeast. This strongly suggests a horizontal transfer of this intron and explains why this intron was placed as an external group in the phylogeny, even though it continued to group with the cox1 introns. In fact, group I introns laterally transferred tend to invade homologous sites in the new host, so it is most probable that this intron 2–3 has come from the cox1 gene from other species [C. gattii species are frequently found on trees, sharing the same habitat with other environmental fungi, such as the species mentioned above. The fact that the greatest similarities and lower e-values were observed between this intron and introns from non-Cryptococcus fungi provides strong evidence for horizontal transfer. However, since there are numerous non-sequenced environmental fungi, it is not possible to precisely determine from which species this intron originated. We can only affirm that it did not come from another Cryptococcus species. Considering the occurrence of such HGT, it is more parsimonious to hypothesize that the position occupied by this intron is a more recent insertion site, as it was absent in all other Cryptococcus genotypes, including another isolate that also belongs to the VGIV genotype. However, in this case, a recent loss could also explain why one VGIV isolate has it and the other does not. Despite being a recent invasion, it can be observed that the branch of this intron is more basal in relation to the other cox1 introns. This can be explained by the fact that it originated from a distant lineage species rather than another Cryptococcus fungus.A new group I intron, not described in the reference sequences, was found occupying a different position in the FC9 isolate (VGIV), between the second and third intronic insertion sites of  species . Studies species . This iscob introns, it can be observed that the group I introns from positions 1 and 2 were more closely related. Overall, the cob and cox1 introns form monophyletic groups, although with the exception of the case discussed above, which confirms that introns from the same gene have more similarities with each other than with introns from other genes. In other words, it is highly likely that an intron has invaded multiple sites within the same gene in the past, prior to the divergence of Cryptococcus species, so that all introns of the same gene share a common ancestor. Moreover, it is noteworthy that in the cladograms presented in cob and cox1 from different Cryptococcus isolates lack informative sites to differentiate among cryptic species. Notably, even the complexes cannot be distinguished, as some introns from C. neoformans are more closely related to certain introns from C. gattii than to other introns from C. neoformans.The phylogenetic analysis was used to uncover similarity relationships among introns from different sites. In the case of mtLSU intronic sequences to our phylogeny , two PCR reactions amplifying different intronic regions . Furthermore, additional tests are still necessary, including more isolates, particularly those from underrepresented genotypes in our fungal collection.The phylogeny showed that the group I introns studied here are not good molecular markers for differentiating genotypes or cryptic species of  regions  can distCryptococcus. This approach is faster and more cost-effective compared to other techniques, such as PCR-RFLP or MLST, commonly used for this purpose, and has great potential for use in research laboratories. Additionally, the data obtained in this study regarding the distribution of group I introns in crucial mitochondrial genes in pathogenic Cryptococcus were at least intriguing, taking into account that cob encodes for cytochrome b, an essential component of the functional unit of Complex III of the electron transport chain, and cox1 encodes for the subunit 1 of cytochrome c oxidase (Complex IV), which plays a role in the final step of the respiratory chain [cob and cox1 products in mitochondria. The data obtained in this study regarding the abundance of group I introns in crucial mitochondrial genes in pathogenic Cryptococcus may serve as a starting point for investigating their impact on mitochondrial gene expression and, consequently, fungal fitness in parasitic conditions.For the first time, a PCR-based method utilizing group I introns has been proposed to differentiate cryptic species in ry chain . The exp


Allium sativum). The nanocomposite obtained is the result of a series of experiments combining the graphene oxide (GrOx) with two members of the Amaryllidaceae family; garlic and onion (Allium cebae), which contain different sulfur materials. The characterization arrays confirmed the successful production of silver crystal, graphene oxidation and the blending of both components. The role of the chitosan as a binder between graphene and silver nanocrystals is proved. Moreover, the study discusses garlic as an optimal source that permits the synthesis of silver nanocrystals (AgNCs) (⁓ 2 to 10 nm) with better thermal and crystallinity properties. It was also confirmed the successful production of the ChAgG nanocomposite. Escherichia coli and Staphylococcus aureus were used to demonstrate the antibacterial bioactivity and L-929 fibroblast cells were utilized to visualize their biocompatibility. The proposed ChAgG nanomaterial will be useful for functionalizing specific fiber network that represents current challenging research in the fabrication of bioactive wound dressings.An adequate wound dressing reduces time of healing, provides cost-effective care, thereby improving patients’ quality life. An antimicrobial bioactivity is always desired, for that reason, the objective of this work is to design an antimicrobial nanocomposite of chitosan/silver nanocrystals/graphene oxide (ChAgG). ChAgG nanostructured composite material is composed of chitosan from corn (Ch), and silver nanocrystals from garlic ( Based on World Health Organization statistics, almost 300,000 people worldwide die from burns each year. In severe burns, the affected cells and blood vessels are often injured and the blood supply to the wound is disturbed. Many factors such as oxygenation, infection, aging, hormones, and nutrition can potentially influence burn progression and disrupt repair with an unbalanced release of various growth factors and cytokines. Different treatment approaches such as dressings and skin substitutes have been applied to aid wound healing. A thorough understanding of the effective factors of burns can improve wound healing outcomes2.The main purpose of wound dressing is to generate a temporary protective physical barrier, absorb wound drainage, and provide the required moisture to optimize re-epithelialization. The choice of dressing depends on the anatomical and pathophysiological characteristics of the wound. Contemporary wound dressings provide additional benefits, such as antimicrobial properties and pain relief3.Despite all, the main issue to solve is bacterial infections in chronic wounds. Electrospun nanofibers offer a promising solution to the management of wound healing, and numerous options are available to load antibacterial compounds onto the nanofiber webs4.The development of new and/or improved antibacterial compound formulations with alternative mechanisms of action is taken into consideration as an important topic to include them in a wound dressing design for overcoming antimicrobial resistance5 demonstrated a strong antimicrobial capacity of a chitosan/silver nanoparticle/graphene oxide nanocomposite using both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. The authors discussed that combining chitosan with graphene oxide promotes interesting properties for its proposal in a wide range of applications, especially in the biomedical field.In 2021, Su et al.6. On the other hand, different carbon nanostructures have been proposed to be used in biomedical applications, mainly graphene10. Currently, different reviews in the literature have reflected these studies, mainly in films of chitosan compounds with oxidized graphene11.Silver nanostructures such as nanocrystals, nanofibers, or nanocrystals possess antimicrobial activity which is useful for its loading in drug delivery systems such as wound dressings and topical gels to treat local wounds12. Chitosan is biodegradable, non-toxic, and promotes wound healing13. Silver on nanometer scales provides antimicrobial properties and reduces inflammation14, and graphene presents antibacterial properties on its own or mixed with different substances. Besides, it has excellent mechanical properties, conductivity, and biocompatibility7.The blend of chitosan, silver nanocrystals, and graphene has been previously investigated demonstrating a great capacity for biomedical applicationsEscherichia coli and Staphylococcus aureus was performed. Finally, a cytotoxicity biocompatibility test using L-929 fibroblast cells were used.Hence, this work aims to prepare and characterize a blend nanocomposite of chitosan, AgNCs, and graphene (ChAgG) and validate its potential biological characteristics to functionalize electrospun fibers for wound dressings. A physicochemical characterization is presented through simultaneous Thermal Analyzer DSC/TGA (SDT), X-ray diffraction (XRD), scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HRTEM), Raman spectroscopy and X-ray photoelectron spectroscopy (XPS) demonstrating the nanocomposite conjugation. To demonstrate the potential use for its incorporation in an electrospun wound dressing, an antibacterial test using Ch) (Merck). Graphene powders (Gr) (LP Bond Research and Development of the Third Millennium S.A. of C.V), and AgNO3 (OMNICHEM) were used as received. Acetic acid at 1% v/v and H2O2 at 30% v/v were used as solvents (Merck). For the synthesis of the nanocrystals (AgNCs), AgNO3 from the commercial company OMNICHEM was used. NH4OH, distilled water, Allium sativum extract (garlic), and Allium cebae (onion) were obtained in the central market of the City of Acatlán de Osorio, México.The antimicrobial nanocomposite ChAgG was prepared using chitosan (Gr) were shredded in a food processor at 600 W for 30 s, they were dispersed at a concentration of 2.41% w/v in hydrogen peroxide (H2O2) at 30% v/v for a period of 30 s. Subsequently, this mixture was placed in a sonic bath at a frequency of 1 Hz for 25 min, until the mixture was homogeneous. The mixture was then placed in a conventional microwave oven for 30 s. After that, the container was removed and placed again in a sonic bath at 1 Hz for 60 s. These last steps were repeated 10 times. Completing these cycles of reactions by microwaves and sonic baths, the temperature increases, and a boiling graphene solution is observed, because of that, it was allowed to stand for 3–4 h cooling to an ambient temperature. To neutralize the H2O2, isopropanol was added in a 1:1 v/v ratio with respect of the H2O2. Finally, a filtering system which includes a Kitasato, Büchner funnel, and a 0.20 µm diameter nitrocellulose membrane was set up to bring the solid residues detained in the membrane, the filtered samples were dried in a conventional oven (FELISA brand) (± 5 °C) at 100 °C obtaining dry graphene. It was stored and labeled in an amber-colored vial until its use.Once the graphene powders (CH3–COOH). To obtain a homogeneous solution, the solution was blended in a food processor at 600 Watts for 30 s and then left to stand at room temperature until there are no more bubbles on the surface.A 2% w/v chitosan solution (Ch), from Merck, was prepared using 1% v/v acetic acid (Allium sativum) (A1–A5) and onion extracts (Allium cebae) (C1–C5), taking into account the base experience by some authors of this research group in the patent previously reported15.In order to obtain a higher yield in the synthesis of silver nanocrystals in our ChAgG nanocomposite, in this research, a series of experiments was performed to find the one with the highest yield, that is, choosing between garlic extracts (H2O) or in ammonium hydroxide (NH4OH), using silver nitrate (AgNO3) in crystal (solid) or in solution (liquid), maintaining in both cases a concentration of 0.98 mmol. To avoid confusing the reader, the TGA and XRD characterization of the samples with the highest concentration of silver nanocrystals between garlic and onion extracts, as well as the HRTEM of the silver nanocrystals used in the ChAgG nanocomposite are presented, where the A5 sample was selected for the synthesis of the ChAgG nanocomposite.Therefore, an experimental design was created where different variables were combined, such as the use of the plant extracts in water -S-methyl-l-cysteine sulfoxide, and γ-l-glutamyl-S-allyl-l-cysteine from garlic were extracted16 as well as thiopropanal sulfoxide, dipropenyl disulfides and propenyl propyl disulfides were obtained from onion17. At the end of this period, the organic solid residues were separated through a cloth mesh by vacuum filtration, and a reflux unit was used  to bring this garlic solution to a temperature of 75 °C. Once this temperature was reached, 1 mmol of AgNO3 was added for 30 min. A color change from turquoise blue to a brick-brown color is observed in the solution, indicating the formation of silver nanocrystals18. After 30 min of reaction at reflux conditions, the oxidized graphene was added in a 2:1 m/m ratio to the AgNCs’s solution, at the same temperature of 75 °C for another 30 min. After this time, the chitosan solution was added at 2% w/v, with an initial concentration of 0.6% v/v to the entire solution, maintaining the temperature reaction at 75 °C for the last 30 min. Finally, a filtering system with Kitasato, Büchner funnel, and 0.20 µm diameter nitrocellulose membrane was assembled and used to obtain the corresponding solids, thus comprising the formation of the graphene nanocomposite, silver nanocrystals, and chitosan (ChAgG compound).Based on the patent application for the synthesis of AgNCsSDT was carried out with a high-resolution TA Instruments 2950 thermogravimetric analyzer. Samples were evaluated in a temperature range from room temperature to 500 °C at a heating rate of 10 °C/ min in air. To perform this analysis of each of the samples, only 2 mL of each sample was taken in liquid, evaporation was not performed because it was possible to obtain results in the liquid state. The liquid samples were stored in a refrigerator (− 4 °C) before being analyzed.Ag crystal structure was confirmed by powder X-ray diffraction (XRD) in a Bruker AXS Model D-B Advance. At room temperature from the range of 5° to 70° for A1 and A5 samples, start: 2.000°–end: 70.000° for ChAgG sample—Step: 0.020°–Step time: 1.2 s–Time Started: 8 s–Cu radiation. The nanocrystals that were infiltrated in the garlic, were subjected to a temperature of 95 °C for 8 days. in an oven (± 5 °C) to perform the evaporation of the solvents and water, and later perform this analysis because of the detachment of the nanocrystals in each of the crystallizers. Any problem was identified at the laboratory temperature and humidity conditions. For the nanocrystals embedded in the onion, the temperature at which they were initially subjected was the same used for garlic, but the detachment of the nanocrystals was not achieved from the crystallizer; so, each of the samples of the onion was subjected to a temperature of 250 °C for 5 days. to achieve detachment of the onion and garlic of the nanocrystals and with this, the unit analysis of the nanocrystals can be performed to determine the crystal structure and morphology.The purity of the reagent and the Allium sativum) was performed by Scanning Electron Microscopy (SEM), on a Leica Stereoscan 440, and High-Resolution Transmission Electron Microscopy (HRTEM) on JEOL equipment model JEM-1200EX. The analysis by SEM required that samples of garlic (Allium sativum) and onion (Allium cebae) were subjected to a solvent evaporation process in an oven at a temperature of 95 °C, for 5 days and then the nanocrystals were removed from the containers . For this analysis HRTEM, the garlic (Allium sativum) and onion (Allium cebae) samples were subjected to the same treatments as in the previous analyzes, but for better evaporation of the solvents, the temperature was adjusted at 400 °C for 3 h. To assemble the samples, a solution with nanocrystals in methanol was made by 10 min shaking in an ultrasound bath. A count of the size of 50 nanocrystals presents in the SEM figure field (50 nm) and 30 nanograins conglomerates in the HRTEM figure (1 µm) were measured, and an average particle <  = change to grain diameters and standard deviations were calculated using the Image J® software.A verification of the nanocrystal morphology on garlic .The XPS apparatus also possesses a μ-FOCUS 500 X-ray monochromator . Binding energies were referenced to the adventitious C1s, O1s, and Ag peaks. Background subtraction was performed using a Shirley baseline, while mixed Gaussian/Lorentzian functions were used to fit core-level spectra.19 and carried out by inoculating the pathogenic strains Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923), previously incubated for 24 h at 35 °C in Mueller–Hinton medium (MHB at 23 g/L) in a 96 microwell plate, where the inoculated medium was placed, adding the sample at a concentration of 10 mg/mL in 0.1% DMSO solvent.ChAgG sample powder was dissolved in 0.1% dimethyl sulfoxide (DMSO) solvent at a concentration of 10 mg/mL. The antimicrobial activity test was performed using the serial dilution methodControls (gentamicin as a positive control and inoculated MHB culture medium as negative control), were handled in parallel to verify the inhibition and normal growth of the pathogens. After the inoculation was completed, the absorbance of each well was read at 620 nm using a microplate spectrophotometer (Thermo Scientific Multiskan). Subsequently, the microplates were incubated at 35° for 24, 48, and 72 h, in order to observe the inhibitory effect on the strains of the samples.A volume of 96 μL of MHB medium was inoculated with the pathogen strain to be tested and deposited in row A of the microplate, to which 4 μL of the sample (at a concentration of 10 mg/mL in 0.1% DMSO solvent) was added and homogenized. From rows B to H, a volume of 50 μL of inoculated MHB medium was deposited. A volume of 50 μL of the homogenate from row A was taken and transferred to row B by homogenizing and completing a total volume of 100 μL, of which a volume of 50 μL was taken, repeating this procedure consecutively until reaching the wells of row H, from which the remaining 50 μL were discarded. Eight dilutions of the sample of interest were obtained from the dilution process , and incubated at 35° for 24, 48, and 72 h, after which the absorbance of the microwell plate was measured at 492 nm, to calculate the inhibitory effect for each of the established concentrations. An inhibition curve was constructed at 24, 48, and 72 h of exposure, separately calculating dose response at these times, which would establish the minimum inhibitory concentration (MIC) for each sample. All experiments were done in triplicate. Statistical analysis (ANOVA P > 0.05) was performed for all groups.mus musculus tissue, were used for culturing. The cells were cultivated in DMEM, supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin–streptomycin, at 37 °C in 5% CO2, until they reached the desired confluency (80%). ChAgG samples were prepared using the eight dilutions  prepared for the antibacterial test. Then, 10, 000 cells were added per well of the L-929 fibroblasts exposing for 24 h to the ChAgG samples under the same conditions of cell incubation. Where diluted phenol 10 mg/mL was used as the positive control and the negative control was the cell suspension without any treatment.The prepared ChAgG sample (10 mg/mL 0.1% DMSO solvent), was also used for the MTT cell viability test. ATCC CCL-1, fibroblast cell line NCTC Clone 929  from After time passed, 10 µL MTT solutions to the 100 µL of the medium in each well were added. The plate was mixed by tapping gently on the side of the tray. Then, it was incubated at 37 °C for 4 h. Subsequently, 200 µL DMSO directly into the medium in each well was added and pipetted up and down several times to dissolve the formazan salt. Finally, the measure of the absorbance signal was made on a spectrophotometer at 570/630 nm. Experiments were done in triplicate. Average absorbance and standard deviation were calculated. Statistical analysis (ANOVA P > 0.05) was performed for all groups.20, TGA also provides important information about the thermal stability and kinetic information of the compounds adding evidence of the incorporation of the silver nanocrystals (AgNCs) to the sample21. The obtention of silver nanostructures can be seen by thermal analysis such as TGA, on account of the high thermal resistance of silver nanocrystals. Despite changes in morphology and size that can be observed with the increment of temperature, silver nanocrystals remain unaltered at high temperatures. When silver nanocrystals are synthesized using a biological source, it is easy to detect because organic matter degrades around 100 °C22.Despite there are several characterization techniques for the determination of the presence of nanometric crystals such as Transmission Electron Microscopy (TEM), X-ray Diffraction (XRD), Atomic Force Microscopy (AFM), Dynamic Laser Scattering (DLS), Small-Angle X-ray Scattering (SAXS), amongst othersNH4OH24 (38–58 °C) and AgNO3 where its operating temperature shift (212–444 °C) due to the presence of all materials. In Figure-It can be seen at the Supplementary Information Figure- of TGA tAgNO3 reagent present at ~ 250 °C, this observed residual mass represented the presence of silver nanocrystals only seen in the garlic source. Hence, it was decided to continue the further synthesis of the silver nanocrystals from the garlic sourceand discard the onion source due to the total discharge of AgNO3, significant residual mass that can compete with the alternative source (garlic).According to what it is seen in Figure-Oscillatoria sp., extract, where they observed a residual mass of 23.46% that was not degraded even at higher temperatures (884–94 °C), endorsing that SDT can be used to demonstrate the presence of silver structures25.Adebayo-Tayo et al. also use the thermogravimetric analysis to monitor the bioproduction of silver nanocrystals using As described in the methodology section, the crystallinity of the silver nanocrystals synthesized from garlic and onion, were compared using the X-Ray Diffraction (XRD) technique. AgNCs samples obtained from garlic presented a higher crystallinity and number of nanocrystals, being A1 and A5 samples the ones with the best results  and Ag (200)27.The graph shows signs which are assigned to A1 and A5 experiments in which more nanocrystals were obtained. In the garlic samples, the presence in the diffraction peaks at 2θ = 38.2° and 44.2° mainly represent crystal planes of Ag, corresponding to (111) and (200)AgNO3 and different solvents was carried out using the X-ray diffraction (XRD) technique. The graph shows signs which are assigned to A1 and A5 experiments in which more nanocrystals were obtained. In the garlic samples, the presence of AgCl as chlorargyrite (PDF 00-001-1238) was observed, presenting a crystalline cubic structure  planes: (120), (102), (022) and (200). Additionally, in red lines Carbon identifying through PDF 00-003-0401, exhibited in d values 3.4000, 2.0600 and 1.6800. As expected, AgNCs are not observed, since they are less than 3%. As it is known to all28 the DRX can detect the materials present in the range of 1–3%, depending on the material analyzed. Continuing with the analysis of the diffractogram as expected, the amorphous part that comprises the nanostructured, organic material and sample holder is observed like a very wide reflection.ChAgG observed in Fig. In another perspective in the diffractogram of the nanocomposite ChAgG, in Fig.  ± 168.2 nm . Samples without graphene oxide showed that the morphology of the AgNCs appears to be mostly spherical, and that they have smaller particle sizes (mean particle size ~ 27.5 nm), and are better dispersed than a group of samples with the presence of graphene oxide where the AgNCs appear to be more agglomerated (raspberry-like) and have a larger particle size (mean particle size ~ 73.69 nm).Su et al.2− in the sensor induced the aggregation of the functionalized AgNCs by selective diazotization coupling reaction in the nanoparticle system30.In another study, silver nanocrystals (AgNCs) using 4-amino-5-hydroxynaphthalene-2,7-disulphonic acid monosodium salt (AHNDMS) and functionalized with p-Aminobenzoic acid (PABA) were prepared, as a sensitive and selective colorimetric sensor for nitrite ions. In that study, SEM results were described, the resulted AgNCs have spherical shape, but, when nitrite ion was introduced with certain concentration to the functionalized AgNCs, the spherical AgNCs changed to an aggregated one, indicating that the presence of the NONigella sativa leaf extract was compared with other chemical synthesis methods for AgNCs. The research group examined their samples trough SEM and particle size analysis that revealed that most of green synthesized AgNCs were spherical in shape with the average size ~ 15 nm and are often agglomerated into small aggregates, while comparing with chemically-synthesized nanocrystals predominately were spherical in shape with the average size 30 nm and were often agglomerated into large aggregates (more than ~ 500 nm)31.Moreover, in a study which a green synthesized AgNCs from E. coli) excellent bioactivity was demonstrated contrary to the S. aureus strain. It is well known that the antimicrobial properties of silver nanocrystals are related to size, whereas lower sizes have better antimicrobial properties32. Despite that, less cytotoxicity in our samples was demonstrated compared with the above studies at similar nanocomposite concentrations.In our study, nanocomposite sizes were bigger than in the previously mentioned studies, and this can be reflected in the antimicrobial properties observed, compared with the cited references, despite those studies which evaluated the antimicrobial bioactivity, different methods than ours were performed, reporting excellent antimicrobial properties. In our study, we do not have excellent results in both bacteria, but for Gram-negative bacteria , the sample used on the ChAgG nanocomposite. The presented micrograph shows the cubic morphology of the obtained nanocrystals in form of crystals, which have diameters in the order of ~ 2 to 10 nm. The calculated nanoparticle average diameter and standard deviation after the measuring of 50 crystals in the field were 5.6 ± 1.5 nm  in face center cubic (fcc), hexagonal close-packed (hcp) and body center cubic (bcc) structures and random compact packaging (rcp). This is due to the collective property, which is due to dipolar interactions or to the order itself (intrinsic), according to what is reported in the literatureIn this study, the spectroscopic characterization  gives evidence of the covalent and ionic interaction of the AgNCs with the oxygen atoms of the oxidized graphene and chitosan discussed in the mentioned sections, these AgNCs are shown in the HRTEM Fig. A.35 prepared through a rapid green synthesis AgNCs using curcumin. In that study, HRTEM was used with the unique purpose in calculating the size of the crystalline nanocrystals, recorded as 12.6 ± 3.8 nm (doubled higher size than our study), which was confirmed by HRTEM, while the face-centered cubic (fcc) crystallographic structure was confirmed by powder x-ray diffraction (PXRD) and selected area (electron) diffraction SAED35.Khan et al.36 discussed the green synthesis of AgNCs using plant sources such as Piper longum, Manilkara zapota, Prosopis juliflora, and Codium capitatum extracts, amongst others. The applications of these AgNCs are intended to be used in biomedical applications such as cancer treatment. As same as our study and Khan’s group35, the HRTEM and SEM were used to observe the morphology and calculate the average sizes of AgNCs (1–100 nm), and crystallographic structure characteristics were analyzed by the XRD36.In another study, Chung et al.36.Comparing the obtained results with the reported literature which synthesized AgNCs from plants, smaller particle sizes but with agglomerated cubic structures were obtained. The resulted nanostructured material presented excellent bioactive propertieshkl values of 0.235 nm and 0.2439 nm for a set of crystal planes at different sites on the AgNCs (8.18 nm of diameter) surface. HRTEM micrographs exhibited well-defined lattice fringes of d111Ag plane . The d-spacing values of the derived diffraction planes from d111Ag = 0.2428 nm and d200Ag = 0.2126 nm, are in good agreement with XRD diffraction pattern of AgNCs39.The XRD pattern of AgNCs presented earlier in Figure-−1, attributed to the G band and the disorder-induced D band, respectively, analogous to those of graphite. On the other hand, the Raman characteristics of graphene-based materials depend not only on the phonon properties but also on the corresponding electronic properties, for example, the linear band structure in single-layer graphene (1LG) and the structures of the different bands. In 1LG, the origin of the harmonic and combining modes, like the 2D modes, define a triple resonance Raman scattering (TRRS) process, which is related to the linear dispersion of its electronic bands. In other words, by keeping this 2D band in mind, we have multilayer graphene, and it appears around ∼ 2700 cm−140. The G band (1580 cm−1) corresponds to vibrations in the graphite plane with the phonon modes in the E2g symmetry  and transverse optics (TO) in the plane). Therefore, the maximum position of the G-mode is sensitive to external perturbations, such as defects, doping, strain, and temperature, and is therefore widely used to probe the responses of graphene-based materials and related devices to external perturbations. In the high-frequency region, due to the weak interlayer coupling in Multilayer Graphene (MLG), the maximum position of the G mode, Pos (G), in the intrinsic N-layer graphene (NLG) is almost 1582 cm−1 and it is not sensitive as such to the number of layers of graphene (N). However, in graphene structures with less than ten layers, they show parameters that depend significantly on the number of layers they have in graphene (N). The D band (1362 cm−1) is generally associated with the respiratory vibrations of the sp2 rings. Therefore, this band is associated with the different disorders that the different NLGs have, which exhibit similar spectral characteristics to the 2D mode. The corresponding fundamental modes of the 2D and D peaks require a surface defect for their activation in Raman scattering. Thus, the absence of the 2D band would give us evidence of the existence of a graphene monolayer of high quality (1LG)41.The strongest and most representative bands in the Raman study of carbon structures are at 1580 and 1362 cm42. On the other hand, to identify the characteristic bands of chitosan and silver nanocrystals, these are indicated based on what is cited in the literature, which indicates that chitosan can be found at 2885 cm−1 and 1654 cm−143, mainly the amine group of chitosan at 1593 cm−144. While AgNCs are concerned, as previously reported, where these nanostructures have decorated chitosan in aerogel, they showed an important interaction in the range of 1000–1800 cm−145, precisely where the ChAgG spectrum widens enormously, overlapping between them, the signals are interacting, corresponding to the D and G bands of graphene, chitosan and silver nanocrystals. This broadening has also been documented when silver nanocrystals interact with graphene bilayers46.According to the results obtained and the tabulated values Fig.  of D, 47.According to the symmetrical shape of the signal identified as 2D, it can be deduced that there is a graphene monolayer, with respect to the 2D/G signal ratio. It can also be concluded that graphene does not have important structural defects until it interacts with AgNCs. However, the increase in the D/G ratio makes it possible to identify surface defects in the oxidation and, at the same time, a disturbance in its lamellar structure when interacting with the silver nanocrystals and chitosan, which when the graphene is oxidized, these oxides are at the extremes of the sheets and that lead to the grafting between the silver nanocrystals and the graphene sheets. Chitosan plays as a binder for the stability of these two nanostructuresC1s) is at 295–281 eV, for oxygen (O1s) is at 540–528 eV and for silver is at 364–376 eV. The three band ranges were evaluated for the nanocomposite ChAgG and, the reference (Gr and GrOx) only C1s and O1s. The deconvolution of the C1s and O1s peaks was based on the common data of different reports on XPS51. For Gr and GrOx , at 287.6 eV for carbonyl (C=O), at 288.6 eV for carboxyl groups (–COOH) of oxidized graphene, at 290 eV for carbonate (C–O*–C=O) and, at 290.9 eV for pi–pi* (π–π*) electronic transitions characteristics in graphene sheets. O1s peaks at 531.2, 532.1, 533.4, 534.3, and 535.5 eV for the molecules of carboxyl (–COOH), carbonyl (O=C), hydroxyl (O–C), carbonate (C–O*–C=O) and water (H2O), respectively.XPS was used to identify the presence of AgNCs in the ChAgG nanocomposite with respect to carbon and oxygen. The XPS spectra of graphene (Gr) and oxidized graphene (GrOx), were recorded as a reference. The range of binding energy for carbon , covalent bond (Ag–O), hydroxyl (O–C), and carbonate (C–O*–C=O), respectively. Finally, there are the two bands that are fingerprints, about the presence of the AgNCs53, this refers to the binding energies of Ag3d5/2 and Ag3d3/2 were 367 eV and 373 eV, respectively.The nanocomposite ChAgG peaks Fig. B are pre51. On the other hand, we can also notice that the hybridization of the defects (sp3) rises with respect to the references, as well as the –OH and carboxyl54. The increase of the π–π* electronic transitions (at 290.9 eV) in oxidized graphene compared to only graphene, suggests that there is exfoliation of the graphene sheets due to peroxide55.Analyzing the percentage arrangement of the peaks described in C1s of the three samples , Supplementary Information Figure-53, It is observed that 50% belongs to an ionic interaction between oxygen-silver, while 21% of this band corresponds to a covalent interaction between these atoms, deducing that the nanocrystals are linked by these two means to oxidized graphene and to molecules of chitosan.To describe the O1s peak of the ChAgG nanocomposite, Supplementary Information Figure-In this study, the Raman and XPS of the chitosan and AgNCs were not performed, since with respect to the Raman assay, only the D/G, and 2D/G bands of the graphene were studied, which are considered fingerprints to demonstrate the grafting on the surface of our nanocomposite. Neither, the XPS of the AgNCs nor chitosan were necessary, because the objective of the XPS is to obtain the presence of silver and its interaction with the oxygen molecules, either from oxidized graphene or of chitosan, for which there is evidence and it has been reported.In this case, it can be said with certainty that the presence of the AgNCs is interacting with the oxygen atoms of the oxidized graphene and chitosan, which together with the other structural characterizations , we have a nanocomposite of graphene, chitosan and silver nanocrystals, called ChAgG.Escherichia coli (ATCC 25922) and Gram-positive Staphylococcus aureus (ATCC 25923) bacteria were selected to evaluate the antibacterial activity of ChAgG samples. In general, the bioactivity of the ChAgG sample differs from the two bacteria tested representing each of their differences in cell wall composition. Nevertheless, the results showed that the ChAgG sample decrease the bacterial population of both bacteria using the highest concentration (400 µg/mL). Still, ChAgG bioactivity was more effective in Escherichia coli (ATCC 25922) than its counterpart Staphylococcus aureus (ATCC 25923).Gram-negative Staphylococcus aureus (ATCC 25923) exposed to ChAgG samples no important reduction (ANOVA P > 0.05) of cell growth was observed, a greater decrease was seen with concentrations of 400 and 200 µg/mL, where approximately a ~ 20% of cell growth reduction was showed. The highest bioactivity observed was with a concentration of 200 µg/mL at 48 h, and 12.5 µg/mL at 72 h, with ~ 75% of cell growth reported. At the lowest concentrations such as 6.25 and 3.125 µg/mL, no alteration in cell proliferation was detected at any time. These results allow us to conclude that, in general, the ChAgG samples were not effective against Staphylococcus aureus (ATCC 25923).In the case of the Escherichia coli (ATCC 25922) showed a more notable antibacterial effect than in the study with Staphylococcus aureus (ATCC 25923), whereas with just 3.125 µg/mL of the ChAgG we observed a pretty interesting bacterial bioactivity reducing bacterial population about ~ 52, 54 and 62% at 24, 48 and 72 h, respectively. The highest bioactivity of ChAgG was detected using 200 µg/mL, at 48 and 72 h, where the % of cell growth was 27 ± 0.5 and 28 ± 1% respectively. Despite good results, our ChAgG samples decrease importantly (ANOVA P > 0.05) the % growth of E. coli (ATCC 25922) but not as high as the gentamicin which was used as a positive control, which reduce the ability of cell proliferation (~ 22%), but optimization of our formulation still can be made increasing its bioactivity.In contrast, Table All data are expressed in % of growth calculated using the growth of negative control (cell growth without any stimuli). Experiments were done in triplicate.Staphylococcus aureus is associated with skin and soft tissue infections in ambulatory care56, while Escherichia coli can be isolated from surgical and traumatic wounds, foot ulcers, and decubitus3. Likewise, each of them are widely studied bacterial models, so they can be used for comparative purposes and likewise as representatives in their cell walls, being E. coli Gram-negative and S. aureus Gram-positive.The selected bacterial strains in this study can cause serious skin damage, for example, Escherichia coli (ATCC 25922), however, against S. aureus (ATCC 25923) was not as effective, a factor to consider is the multiple drugs resistance phenotype of the S. aureus bacteria (ATCC 25923). That makes it one of the most difficult pathogenic bacteria to treat57, so one of the reasons to which can be attributed these results may be the low percentage of the antibacterial compound used. Hence, Ch-functionalized compound show higher efficacy for E. coli (ATCC 25922) than for S. aureus (ATCC 25923) due to the difference in their cell walls in a study published by Dutta et al., in which it has also been shown that the antibacterial activity of chitosan incorporated, either with silver ions (Ag) or nanocrystals, is greater than the activity of each component individually58.The results showed that ChAgG compound has better behavior against E. coli (ATCC 25922). Similar results were reported by Gu et al., where it was evaluated the antibacterial properties of graphene supplemented with chitosan, confirming that the inhibition effect was stronger and dependent on the concentration of said components59.It has also been shown that the potential of G-based bacterial surfaces can be enhanced by using silver, zinc, and iron nanocrystals. Our ChAgG sample, as already mentioned, showed greater efficacy against 60. In this case, it was carried out with mouse fibroblasts L-929 where after 24 h of incubation it was observed that there was no difference between cell proliferation when compared to control .The MTT assay was performed to measure the metabolic activity of cells and estimated through the ‘color-change’ phenomenon from yellow-colored tetrazolium salt, MTT 3--2,5-diphenyltetrazolium bromide to purple-colored formazanThe proliferation of the L-929 cultures showed that the ChAgG compound decreased by ~ 21%. A slight pattern can be observed where between higher the concentration of the ChAgG compound higher the alteration of cell growth (%). Nevertheless, at 100 µg/mL was observed the highest cytotoxicity with a ~ 16% of cell growth reduction, and at 3.125 µg/mL a slight increment of 2% was observed. Almost no alteration (ANOVA P > 0.05) in cell growth was observed between concentrations of 50–3.125 µg/mL. Despite that, all these positive or negative alterations do not represent a major change in cell behavior.When performing an analysis of variance (ANOVA P > 0.05) comparing the unexposed samples with those exposed for 24 h, it was observed that there was no significant difference in the normal growth of the L929 fibroblasts. Although, in the analysis performed comparing the several concentrations between them, they did not show a significant difference to decide which of the samples is better, however, by showing that there was no significant alteration in the normal growth of L929 fibroblasts after 24 h, we assume that the ChAgG compound is not toxic to the human body, but this biocompatibility will depend of concentration, where at highest concentration cytotoxicity can be increased Fig. .Figure 661.L929 cells were used in the present study because they can be easily cultured in a reproducible manner, and also this cell line is widely used for preliminary cytotoxicity evaluation for a wide range of biomaterials because of its easy proliferation and adherence on most of the biomaterial surface62. Moreover, Toullec et al. demonstrated that the presence of pure chitosan electrospun scaffolds does not provoke any alteration on L929 cell proliferation63.About the reported cytotoxicity of chitosan, silver nanocrystal, and graphene in the presence of L929 cells. Gao et al. discussed the good biocompatibility and improved cellular attachment of L929 fibroblast cells64 evaluated the cytotoxicity of pristine graphene monolayer using the murine fibroblast L929 cell line. In that study, graphene demonstrated no cytotoxicity in L929 fibroblast and increased cell adhesion and proliferation within 24 h of culture. The authors discussed the potential capacity of graphene for the recovery of damaged subcutaneous connective tissue after injury64.In the case of the cytotoxicity of graphene, Lasocka et al.65. Hence ChAgG sample’s biocompatibility will depend on the concentration of AgNCs present in the final formulation.Finally, AgNCs cytotoxicity on L929 cells is concentration-depended, hence, a higher concentration of AgNCs presence lower cell proliferation, this phenomenon is observed in our ChAgG samples as well as the study of Składanowski et al., where the patterns of the same results are observed between both studiesThe objective of this study is to report a sequential set of characterization techniques that helps in the decision making of the experimental steps for the antimicrobial ChAgG nanocomposite synthesis. From the selection of a natural source for AgNCs production to the demonstration of chemical interaction between chitosan, graphene oxide, and the resulting AgNCs. Table From an initial screening of 10 samples, 5 using garlic as a source (A1–A5) and 5 using onion (C1–C5), were tested and the residual mass percentage (Ag presence) was calculated selecting two samples from the garlic source. Secondly, the cubic structure of the resulting AgNCs was characterized with XRD and general morphologies, and the size of the AgNCs nanocrystals and agglomerated were tested with HRTEM, and SEM, respectively. Then, Raman and XPS demonstrated the chemical interaction between chitosan, graphene oxide, and AgNC. Finally, bioactivity and biocompatibility were successfully demonstrated.66.Traditional wound dressings e.g., bandages and gauzes, although highly absorbent and effective for drying mild, exuding wounds, require a regular application, which therefore can cause pain upon dressing change. In addition, they have poor adhesion properties and cannot provide enough drainage for the wound. In this regard, the normalization of the healing process in chronic wounds is an extremely urgent task of public health and requires the creation and implementation of affordable dressings for patients with chronic wounds67.Despite all, the main issue to solve is bacterial infections in chronic wounds. Electrospun nanofibers offer a promising solution to the management of wound healing, and numerous options are available to load antibacterial compounds onto the nanofiber webs2O3 fibrous scaffolds are non-toxic, flexible, with antimicrobial activity , and can absorb and retain water. These components were effectively to be used as wound dressing generating effective wound healing. The biocompatibility was evaluated in HFF-1 fibroblast after 24 h of exposure69.In our previous work, it was demonstrated that PCL/PVP 85:15 and PCL/PVP loaded with Ag-Si/Al12. Chitosan is biodegradable, non-toxic, and promotes wound healing13, silver nanocrystals (AgNCs) give antimicrobial properties and reduce inflammation14, and graphene also confers self-antibacterial property and antibacterial properties in combination with different substances, excellent mechanical properties, conductivity, and biocompatibility7.The blend of chitosan, silver nanocrystals, and graphene has been previously investigated demonstrating a great capacity for biomedical applicationsHence, our research group will prepare and characterize PCL/PVP-ChAgG fibers and test their morphological, physicochemical, and mechanical properties of the fibers to create potential wound dressing for further biological studies.Escherichia coli rather than Staphylococcus aureus and the final composite does not provoke important cytotoxicity to L-929 fibroblast cells validating its biocompatibility in vitro. For future work, the ChAgG nanocomposite will be used to functionalize electrospun nanofibers to create bioactive antimicrobial wound dressings.Bioactive wound dressings to treat superficial skin or burn wounds that generate high exudation are desired. One of the important issues in these treatments is microbial contamination and dressing attachment to the affected zones, complicating skin healing and recovery. Our work proposed an antimicrobial nanocomposite (ChAgG) that includes chitosan, silver nanocrystal, and graphene. These components will give the final wound dressing a potential bioactive characteristic. The characterization arrays which include TGA, XPS, Raman, HRTEM, and TEM demonstrated the successful production of silver crystal graphene oxidation and the blending of the components. TGA was useful to decide whether silver nanocrystals will be prepared from garlic. X-ray diffraction demonstrated that A5’s sample has a higher crystallinity with respect to A1’s sample, therefore, being A5 the ideal preparation for the synthesis of the ChAgG nanocomposite. HRTEM and TEM demonstrated the silver nanocrystal formation with cubic structures with sizes of 2–10 nm, Moreover, the Raman study provides evidence that chitosan plays a binder role between graphene and silver nanocrystals. XPS demonstrated the successful preparation of the whole ChAgG nanocomposite. Finally, it was demonstrated that the ChAgG composite possesses better antimicrobial bioactivity against Supplementary Information.


TAR DNA-binding protein 43 (TDP-43) is a versatile RNA/DNA-binding protein with multifaceted processes. While TDP-43 has been extensively studied in the context of degenerative diseases, recent evidence has also highlighted its crucial involvement in diverse life processes beyond neurodegeneration. Here, we mainly reviewed the function of TDP-43 in non-neurodegenerative physiological and pathological processes, including spermatogenesis, embryonic development, mammary gland development, tumor formation, and viral infection, highlighting its importance as a key regulatory factor for the maintenance of normal functions throughout life. TDP-43 exhibits diverse and sometimes opposite functionality across different cell types through various mechanisms, and its roles can shift at distinct stages within the same biological system. Consequently, TDP-43 operates in both a context-dependent and a stage-specific manner in response to a variety of internal and external stimuli.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-023-01336-5. TARDBP gene, TDP-43 is highly conserved across various species . Additionally, TDP-43 exhibits a dual role in the complex regulation of cancer-related miRNAs, acting as a promoter of cancer progression through the regulation of miR-423-3p, while also exerting inhibitory effects on cancer progression through the regulation of miR-500a-3p [TDP-43 also affects diverse functions of downstream genes. In HeLa cells, TDP-43 inhibits CDK6 by recruiting UG-rich nscripts , whereas-500a-3p .These studies underscore the heterogeneity of cancer and highlight the intricate regulatory role of TDP-43 in cancer progression. Although the mechanisms by which TDP-43 regulates cancer are complex and not yet fully understood, they likely depend on the specific cellular context and cancer type.Accumulating evidence suggests the existence of an inverse comorbidity phenomenon between oncological and neurodegenerative conditions. In 2014, Ibanez et al. conducted a transcriptomic meta-analysis of several neurodegenerative diseases and three cancers . They discovered a significant overlap between up-regulated genes in neurodegenerative diseases and down-regulated genes in cancer, as well as down-regulated genes in neurodegenerative diseases and up-regulated genes in cancer . SubsequFurthermore, the intricate mechanisms underlying the effects of TDP-43 in neurodegenerative diseases can be elucidated from various perspectives, including point mutations within the genome, multiple splicing mutants, and phosphorylation proteins. However, it remains to be investigated whether TDP-43 mutations and truncations exist and hold significant roles in cancer. Additionally, given its role as a splicing factor, the phosphorylation status of TDP-43 directly impacts its splicing function, prompting inquiry into the presence of abnormal phosphorylation of TDP-43 in tumor cells. While exogenous overexpression of TDP-43 can lead to elevated expression of the 35 kD isoform, which may exert cytotoxic effects on cancer cells and induce cell death , furtherTDP-43 is expressed in almost all cell types and is highly conserved among different species. Increasing evidence confirms its crucial regulatory role in various developmental processes, including embryonic development, body fat metabolism, mammary gland development, the reproductive system and so on.Tardbp mRNA [in vitro, blastocysts with homozygous Tardbp deletion exhibit impaired outgrowth in the inner cell mass. Conversely, mice with heterozygous TDP-43 deletion display normal development and fertility, with no noticeable abnormalities up to 14 months of age and no changes in TDP-43 protein expression compared to wild-type mice [Tardbp knockout mice died between ED 3.5 and 8.5, heterozygous Tardbp mice showed no differences in TDP-43 expression or phenotypic changes, including body weight, growth rate, appearance, and fertility, and further displayed no gross tissue abnormalities up to the age of 6 months compared to control littermates [Tardbp mRNA and produced a nonfunctional truncated TDP-43 variant in mice after crossing with the CAG-Cre transgenic mouse line [Various studies utilizing knockout mice have provided compelling evidence for the crucial roles of TDP-43 in embryonic development. Researchers have previously constructed TDP-43 knockout mice by targeting the translation initiation site of exon 2 of dbp mRNA , with suype mice  also repuse line . In accoTardbp knockout mice remained stable across these models, indicating that TDP-43 possesses an inherent regulatory mechanism to mitigate the adverse effects of TDP-43 deletion.Collectively, these different mouse models demonstrate that homozygous loss of TDP-43 can result in embryo death during blastocyst implantation, confirming the necessity of TDP-43 expression in embryonic development. In addition, protein levels in heterozygous Regarding the mechanism by which TDP-43 safeguards embryonic development, a recent study conducted in mouse embryonic stem cells (mESCs) discovered that TDP-43 protects the embryonic genome by interacting with L1 open reading frame 1 protein (L1 ORF1p) .Tardbp mice with Rosa26-ErCre mice (which express Cre protein upon tamoxifen induction) to generate inducible Tardbp knockout mice. Upon tamoxifen induction, these conditional homozygous Tardbp knockout mice unexpectedly died by day 9, characterized by substantial fat loss and increased fatty acid consumption. Mechanistically, deletion of TDP-43 down-regulated the expression of obesity-associated gene Tbc1d1, leading to alterations in body fat metabolism [In addition to its vital role in embryonic development, the potential functions of TDP-43 in adult animals have been increasingly investigated. To overcome the issue of embryonic death caused by TDP-43 deletion, researchers have employed conditional knockout mice to study the physiological function of TDP-43. For example, Chiang et al. crossed floxed A315T mutant mice (ALS model) can restore bioenergetic balance and extend lifespan [Cyp8b1 by binding with the lncRNA lncLSTR, thus regulating systemic lipid metabolism in mice [Accumulating evidence supports the involvement of TDP-43 in fat metabolism. Stallings et al. demonstrated that TDP-43 regulates fat homeostasis and glucose metabolism . Coughlalifespan . Egawa elifespan . Li et a in mice . Studies in mice . In our Lipids serve as essential components of biological membranes, not only providing efficient energy storage for organisms, but also acting as signaling molecules/messengers in diverse developmental pathways , with thAcrv1, which codes for the sperm acrosomal protein SP-10, is strictly expressed at the transcriptional level in the testes and round spermatids. Evidence has shown that the proximal promoter of Acrv1 is sufficient to maintain round spermatid-specific expression and acts as an insulator, preventing ectopic expression of Acrv1 in somatic cells [Acrv1 promoter via two GTGTGT-motifs, eventually inhibiting premature Acrv1 expression during spermatogenesis [Acrv1 gene transcription in a histone deacetylase-independent manner [Acrv1 ectopic expression [Precise regulation of cell type-specific gene transcription is crucial for spermatogenesis, where a multitude of testis-related genes are activated in a programmed spatiotemporal order. For example, ic cells . TDP-43 ogenesis . TDP-43 t manner . In somapression . These fSubsequent investigations have revealed that TDP-43 is expressed in both Sertoli cells and germ cells, indicating its potential adoption of multiple conformational states during different stages of spermatogenesis. Studies have shown that TDP-43 expression initiates in type B/intermediate spermatogonia, reaches its peak in preleptotene spermatocytes, disappears in leptotene and zygotene spermatocytes, reappears in pachytene spermatocytes and early round spermatids, and subsequently decreases in later spermatids . The varLactation, a highly distinctive feature of mammals, provides offspring with sufficient nutrition to ensure their survival. In earlier research, we found that TDP-43 is required for mammary gland development , 83.In our previous studies focused on identifying key post-transcriptional regulatory genes during lactation and exploring the impact of positive selection on species divergence, we conducted an evolutionary analysis of RNA-binding proteins (RBPs) across 15 mammalian species. Our analysis revealed that TDP-43 underwent positive selection within the mammalian lineage, after applying false discovery rate correction. Subsequently, we investigated the expression patterns of TDP-43 during different stages of mammary gland development in mice and observed high expression levels during pregnancy and early lactation. Notably, TDP-43 knockout in the mouse mammary gland resulted in a significant reduction in milk production, with subsequent starvation of the pups within three weeks postpartum. Furthermore, we collected human milk from lactating women and analyzed the expression level of TDP-43 in isolated RNA from the milk lipids. Remarkably, we found a positive correlation between TDP-43 expression and higher milk output, suggesting that TDP-43 mRNA expression may serve as a potential indicator of lactation in parturient women . FurtherSox2 alternative polyadenylation. Additionally, TDP-43 is also reported to regulate the expression of the Neat1 gene, repressing the formation of paraspeckles. Notably, during differentiation, TDP-43 is recruited to paraspeckles and facilitates the exit of ESCs from pluripotency and embryonic patterning [Stem cells are necessary for tissue development, homeostasis, and repair . Abnormatterning . Moreovetterning .Myolinc and regulates muscle-related gene expression, while knockdown of TDP-43 inhibits myogenic differentiation [TDP-43 is also implicated in various other developmental processes. Regarding myogenesis, Militello et al. found that TDP-43 binds to the muscle-enriched lncRNA ntiation . Furtherntiation . Li et antiation . In the ntiation . TDP-43 ntiation .Initially recognized as a protein associated with HIV, TDP-43 has been increasing implicated in the entry, replication, and latency of other viruses  beyond HHIV-1 gene expression [Malat1 and undergoes cleavage to form the TDP-35 isoform. This process promotes the production of IRF3-initiated antiviral type I interferons (IFNs) by preventing IRF3 proteasomal degradation, thereby enhancing host defense against viral infection [Multiple studies have provided evidence that TDP-43 acts as a protective factor in individuals during viral infections. Ou et al. first cloned and characterized TDP-43 in 1995, demonstrating its binding to the TAR sequence motifs of HIV-1 and inhibition of pression . Althougpression , more repression , 95. Addnfection .TDP-43 may also have negative effects on individuals. For example, during HBV infection, TDP-43 acts as a host factor that stimulates gene transcription by binding to the HBV core promoter, forms complexes with other proteins supporting the HBV life cycle, and inhibits pregenomic HBV RNA splicing, thereby promoting the production of HBV replicative intermediates, mRNAs, proteins, and virions .The above findings highlight the wide-ranging roles of TDP-43 throughout various stages of mammalian life, encompassing early development to aging  technology has revealed that TDP-43 can bind with more than 6 000 mRNA targets, representing about 30% of the entire transcriptome , 115, thchanisms . FurtherTDP-43 serves a critical function in the cellular stress response. Notably, the TDP-43 protein can sense intracellular signals, such as misfolding proteins Sup35, Pab1, and Pub1 . In ovarThe above studies emphasize the diverse and sometimes opposing functions of TDP-43 in various tissues and developmental stages, which may be attributed to the following factors:


Transplant recipients with CMV infection have a high risk of complications and mortality. This study aimed to better understand patient and CP knowledge of post-transplant CMV and identify how it can be improved.Fig 1). We report results from Phase 2 (September 2022–March 2023), which included an educational webinar with a pre- and post-webinar assessment comprising 4 questions on the risk, symptoms, treatment, and complications of CMV; participants (pts) self-reported their confidence rating pre- and post-webinar. Transplant recipients and CPs also completed a quantitative survey on CMV management and patient/CP burden. Pts were recruited via the PatientsLikeMe (PLM) website, email, or social media.The study included 2 research phases (Table 1). Patients primarily contacted their surgeon about CMV and sought information on the internet.The webinar was completed by 33 pts. There was a total of 86 and 121 correct responses to the 4 pre- and post-survey questions, respectively, indicating a significant improvement in understanding of CMV post-webinar as compared with before . Almost half (n=16 [48%]) of pts reported feeling “not at all confident” in recognizing CMV symptoms post-transplant pre-webinar; post-webinar, this was 0 pts. Twenty-eight transplant recipients  and 1 CP completed the quantitative survey. Half of recipients received a CMV diagnosis post-transplant and 16/28 reported having no awareness of their or their donor’s CMV status prior to transplant. Most (27/28) recipients reported a positive experience with their care team post-transplant. Half of recipients set goals with their care team or were provided resources to track them post-transplant. Recipients reported CMV medication changes and challenges with treatment (Although transplant recipients reported positive experiences with their healthcare team, the post-transplant care journey could be improved by better educating patients about CMV and CMV management. Aggregate-level learnings from the study with be shared on the PLM platform to reinforce the value of participation in research.Funding: Collaborative research study; primary funding from Takeda Pharmaceuticals U.S.A., Inc.Deepshikha Singh, MPH, PatientsLikeMe: Employee|PatientsLikeMe: Ownership Interest Mary C. Burke, MHA, PatientsLikeMe: Employee|PatientsLikeMe: Ownership Interest Subhara Raveendran, PhD, Ascendis Pharma: Employee|PatientsLikeMe: Ownership Interest Erlyn Rachelle Macarayan, PhD, PatientsLikeMe: Employee|PatientsLikeMe: Ownership Interest Maisha Razzaque, MS, PatientsLikeMe: Employee|PatientsLikeMe: Ownership Interest Genovefa Papanicolaou, MD, Allovir: Advisor/Consultant|Amplyx: Advisor/Consultant|Astellas: Advisor/Consultant|Cidara: Advisor/Consultant|CSL Behring: Advisor/Consultant|DSMC: Advisor/Consultant|Merck: Advisor/Consultant|Merck: Grant/Research Support|Merck: institutional research support for clinical trials|MSD: Advisor/Consultant|Octapharma: Advisor/Consultant|Partners Rx: Advisor/Consultant|Shire/Takeda: institutional research support for clinical trials|Symbio: Advisor/Consultant|Symbio: Advisor/Consultant|Takeda: Advisor/Consultant|Vera Pharma: Advisor/Consultant Macey L. Levan, PhD, Takeda Pharmaceuticals: Advisor/Consultant Maggie McCue, MS, RD, Takeda Pharmaceuticals U.S.A., Inc.: Employee Megan Gower, PharmD, Takeda Pharmaceuticals U.S.A., Inc.: Employee|Takeda Pharmaceuticals U.S.A., Inc.: Ownership Interest Daniele K. Gelone, PharmD, Abbott Laboratories: Ownership Interest|Ionis Pharmaceuticals: Ownership Interest|Johnson & Johnson: Ownership Interest|NovoNordisk: Ownership Interest|Pfizer: Ownership Interest|Regenxbio: Ownership Interest|Takeda Pharmaceuticals U.S.A., Inc.: Employee|Takeda Pharmaceuticals U.S.A., Inc.: Ownership Interest|Vertex Pharmaceuticals: Ownership Interest