Staphylococcus aureus isolates. The UA-Ch-NPs were synthesized by a simple method and then characterized by TEM, FTIR, DLS-zeta potential, and XRD analyses. According to the characterization results, highly dispersed spherical nanoparticles with a mean diameter of 258 nm and a zeta potential of + 40.1 mV were developed. The antibacterial properties of UA-Ch-NPs were investigated and their inhibitory effect on biofilm formation was demonstrated by AFM. Finally, the expression levels of icaA and icaD were measured using real-time PCR. Results indicated that the minimum inhibitory concentration (MIC) of UA and UA-Ch-NPs against S. aureus was 64 and 32 µg/mL, respectively. The treatment of bacterial cells with UA-Ch-NPs significantly decreased the expression of icaA and icaD genes which are engaged in biofilm formation. Our results indicated that UA-Ch-NPs could be a promising material for antibacterial and antibiofilm applications.  The present study aimed to synthesize ursolic acid-loaded chitosan nanoparticles (UA-Ch-NPs) as an antiinfective agent against 21  Staphylococcus aureus as a Gram-positive bacterium causes several infections including nosocomial infections. S. aureus infections have been shown to be highly variable. For instance, S. aureus causes acute diseases, like bacteremia and severe chronic infections [1]. In recent years, S. aureus has represented a serious challenge because of its ability for developing resistance against currently available antibiotics. This resistance is generally linked to the capability of the bacteria to form biofilm. S. aureus strains that are isolated from mastitis in ruminants, for example, produce slime in vivo, resulting in a remarkably higher capacity of colonization in comparison with nonslime producing variants. As a result of biofilm formation in mastitis isolates, susceptibility to antibiotics is reduced, which is the consequence of the reduced antibiotic diffusion through the biofilm matrix and a reduction of the metabolic activity of bacteria inside the biofilm [2]. Thus, a new generation of antibacterial agents is urgently needed to fight S. aureus.E. coli and S. aureus . UA has been indicated to have various effects on some microorganisms, characterizing it as an antibiotic drug. Kozai et al. [5] demonstrated that UA managed to inhibit insoluble glucan synthesis in Streptococcus mutans. Moreover, UA has been shown to fight against S. sobrinus and S. mutans, with a minimum inhibitory concentration (MIC) of 2.0 μg/mL [6]. Furthermore, the antibacterial properties of UA have been discovered on other human bacterial pathogens, including S. pneumonia, methicillin-sensitive, and methicillin-resistant Bacillus cereus, Staphylococcus aureus, and Pseudomonas aeruginosa [7]. Ursolic acid  as a natural pentacyclic triterpenoid carboxylic acid is found in various plants. This agent is highly potential to provide defense against certain pathogens such as Despite its ability to kill bacteria, UA has low water solubility which limits its clinical applications. Therefore, different methods were proposed for improving UA antibacterial activity. Some nano-drug delivery systems have been used for increasing drug solubility . In this regard, some natural water-soluble polymers, like alginate, chitosan, or poly(lactic-co-glycolic acid) have been widely employed in drug delivery systems owing to their biodegradability and biocompatibility features [10–13]. S. aureus. It was assumed that UA-Ch-NPs could overcome the solubility of UA and improve its efficacy. The biological applications, such as growth-inhibitory effects and biofilm formation prevention activity of this agent were evaluated as well.Chitosan (Ch) is a natural polysaccharide polymer; it is a deacetylated chitin derivative. Thanks to its low toxicity and high biocompatibility, this biological material has been widely used in many biomedical applications . Ch has potency for drug delivery through the paracellular pathway. Jin et al. [10] loaded UA in Ch nanoparticles to study their antiangiogenesis activity in tumors through in vitro and in vivo investigations. Although synthesis of UA-loaded Ch nanoparticles and their antitumor properties have been reported, to the best of authors’ knowledge, there is no study concentrating on the antiinfective properties of the UA-loaded Ch nanoparticles. Therefore, this study aims to investigate the antiinfective properties of ursolic acid-loaded chitosan nanoparticles (UA-Ch-NPs) against n = 1600 determined by the supplier) and ursolic acid (98.6%) were purchased from Sigma-Aldrich Co., Germany. Ethyl-(3-3-dimethylaminopropyl) and N-hydroxy-succinimide (NHS) were purchased from Beyotime Institute of Biotechnology, China. S. aureus ATCC 25923 was supplied by Pasteur Institute, Iran. Penicillin and Ciprofloxacin were obtained from Jaber Ebne Hayyan Pharmaceutical Co., Iran. Ceftiofur, erythromycin, tetracycline, trimethoprim, ampicillin, chloramphenicol, and gentamicin were obtained from Pharmacia & Upjohn, Belgium.Chitosan , and 8 mg N-hydroxysuccinimide (NHS) were mixed with the obtained solution. It was then vortexed overnight at ambient temperature. Then, the sample was centrifuged for 20 min at 11,000 rpm. The harvested pellets were UA-Ch-NPs that were lyophilized for prolonged storage.To confirm the successful production of UA-Ch-NPs, Fourier transform infrared (FTIR) analysis was conducted using a FTIR spectrophotometer (Perkin Elmer Spectrum RXI). This experiment was carried out for UA, Ch, and UA-Ch-NPs samples. Transmission electron microscopy  was used to study the morphology of the prepared samples and Digimizer image analysis software (version 5.3.4) was used to measure the mean size of the nanoparticles. The X-ray diffraction (XRD) analysis was also performed using an INEL Equinox 3000 diffractometer with the CuKα (λ = 1.5406 Å) radiation. The size and surface charge of the produced UA-Ch-NPs were analyzed using DLS-zeta potential analyzer . The surface zeta potential of UA-Ch-NPs was calculated using a dynamic light scattering (DLS) instrument (Brookhaven 90 Plus/BI-MAS). S. aureus ATCC 25923 was purchased from Pasteur Institute of Iran. A total of 21 S. aureus isolates obtained from bovine mastitis were used in this study; the isolation was carried out in the pharmaceutical laboratory, Shahid Beheshti University of Medical Sciences, Tehran, Iran. The disc diffusion approach was used to carry out antibiotic sensitivity testing (AST) against ceftiofur, erythromycin, tetracycline, trimethoprim, penicillin, ampicillin, chloramphenicol, gentamicin, and ciprofloxacin. Firstly, inoculation of the Mueller–Hinton agar Petri dishes with half-McFarland of bacteria was done, followed by the placement of antibiotic discs right onto the agar. The diameters around the discs were determined after 24 h of incubation. The sensitivity to UA-Ch-NPs was assessed based on the inhibition zone diameter, based on the Clinical and Laboratory Standards Institute (CLSI) protocol [16].The MIC value of all pharmaceutical agents was measured by microdilution assay in 96-well plates. Accordingly, 100 µL of sterilized Mueller–Hinton broth (MHB) was added to each well, followed by treating each well separately with serial dilutions of UA and UA-Ch-NPs. The microbial cultures were then adjusted to the turbidity of a 0.5 McFarland standard. The 0.2 mg/mL p-iodonitrotetrazolium chloride (INT) was mixed with each sample. Finally, following incubation at 37 °C for 24 h, the lowest concentration before the color change was determined as the MIC value. 600). The bacteria adhesion to the oil phase was quantified through calculation of the ratio between the optical density prior to and after mixing. Finally, the hydrophobicity degree was categorized into three classes: > 70%, 30%–70%, and < 30% for strong, moderate, and low hydrophobicity, respectively. The bacterial adhesion to hydrocarbons test was used for measuring the relative hydrophobicity of various bacterial strains following the work by Subbiahdoss et al. [17]. In short, bacterial suspension was mixed with n-decane (hydrocarbon phase) at a 1:1.2 ratio, followed by vortexing for 2 h. After the separation of the obtained emulsion, the optical density of the aqueous phase was measured at 600 nm  supplemented with 1% glucose and then seeded into 96-well flat-bottom sterile polystyrene microplate. The broth without bacteria and the inoculated broth without the nanoparticles were considered as negative and positive controls, respectively. The samples were incubated at 37 °C for 24 h. Biofilm stains and the culture medium were removed and washed with phosphate buffered saline (PBS). Afterward, 100 μL gentian violet, 1%, was added and incubated at room temperature for 5 min. The samples were then washed with distilled water to wipe up the excess gentian violet. Eventually, 200 μL glacial acetic acid (33% (w/v)) was added to each well. After 20 min, the ELISA reader  was used to read the absorbance of each sample at 640 nm [18]. 640 values were employed for comparative analysis and semi-quantitative classification of the biofilms produced by the bacterial strains based on the approach presented by Stepanovic et al. [19]. Briefly, the cut-off OD (ODc) was set as three standard deviations above the mean OD of the negative control, and classification of strains was as follows: OD > 4 × ODc = strong biofilm producer; 2 × ODc < OD < 4 × ODc = moderate biofilm producer; ODc < OD < 2 × ODc = weak biofilm producer; and OD < ODc = poor biofilm producer.The OD2. The silicon nitride tips used in the measurements were irradiated with UV for 15 min in the air to remove any organic contaminants. The tip curvature radius was less than 10 nm. The force constant and oscillation frequency were 0.03 N/m and 255 kHz, respectively.Atomic force microscopy  was used to study the biofilm formation in a contact mode of 10 × 10 µm620 values of bacterial cultures were measured at 15 min intervals for 24 h and each one was read 3 times. OD measurements for the development of bacterial growth curves were performed by determining the absorbance at a wavelength of 620 nm. Bacteria were cultured in Costar 96-well flat-bottom plate and treated with different concentrations of UA and UA-Ch-NPs . The ODS. aureus using commercial RNA extraction kits  according to manufacturer instructions. The equal quantity of RNA extracted from each group of bacteria was treated by 5 μL of DNase 1  for the probable presence of contaminating DNA. The RNA samples were then checked for the 16S rRNA gene as an internal standard using below 16S primers. According to the manufacturing protocol , total RNA (2 μg) was used for cDNA synthesis. The reaction was carried out under the following conditions: one cycle at 37 °C for 15 min for reverse transcription, 94 °C for 30 s for the inactivation of enzymes up to 10 μL, and without hot commencement [20]. The RT-qPCR reactions were performed using SYBR Green PCR Master Mix  and ABI 7000 instrument . The used primers were as follows: icaA Forward; 5-ACA CTT GCT GGC GCA GTC AA-3, icaA Reverse; 5-TCT GGA ACC AAC ATC CAA CA-3, icaD Forward; 5-ATG GTC AAG CCC AGA CAG AG-3, icaD Reverse; 5-TGA ACT TAT TCC ACC GCC TTT A-3, Bap Forward; 5- ATA CTG ATG GCG ATG GTA-3, Bap Reverse; 5-ACT GTG TCT TCT GTT GTA AT-3, 16S forward; 5-ACT GGG ATA ACT TCG GGA AA-3 and 16s reverse; 5-CGT TGC CTT GGT AAG CC-3. The PCR mixture’s final volume was 25 μL with 12.5 μL of the master mix, 1 μL each of forward primer and reverse primer, 1 μL of cDNA, and 9.5 μL of free water. The cycling conditions were: 1 cycle at 95 °C for 10 min, and then 40 cycles at 95 °C for 20 s, and 60 °C for 40 s. Finally, to draw the melting curve, the samples were incubated at 95 °C for 15 s, 60 °C for 30 s, and 95 °C for 15 s.Total RNA was extracted from treated and untreated One-way ANOVA was used for the statistical analysis of the experimental data, and results were presented as means ± SD. Also, p < 0.05 was considered statistically significant to verify the variations in the results due to the effect of different treatments. –1 is observed, which is identified as the O–H stretching vibration. The peak at 2940 cm–1 corresponds to the C-H stretching vibrations in methylene groups. The bands at 1720, 1380, 1463, and 1090 cm–1 are related to the C=O stretching vibrations in the carboxylic acid group, C-H groups, C=C stretching vibrations, and C-O stretching vibrations in secondary alcohols, respectively [21]. Ch also showed some characteristic peaks in the FTIR analysis. The presence of O-H, N-H, and intramolecular hydrogen bonds in Ch was confirmed by stretching vibrations around 3200–3600 cm–1. The peaks at 2920 and 2870 cm–1 are related to symmetric and asymmetric stretching vibrations of C-H groups, respectively. The bands at 1650, 1590, and 1350 cm–1 are associated with the C=O stretching vibrations in type I amide, the N-H bending vibrations in type II amide, and the C-N stretching vibrations in type III amide, respectively. The peaks at 1450 and 1360 cm–1 are attributed to the C-H bending and symmetrical vibrations in CH2 and CH3. The absorption bands at 1150 and 1000 cm–1 are related to asymmetric vibrations of C-O-C bridges and C-O stretches, respectively . The changes in the intensity of these peaks and the formation of new peaks in the FTIR spectrum of UA-Ch-NPs suggest the functional interaction between UA and Ch. Two small peaks at 1600 and 1700 cm–1 in the spectrum of UA-Ch-NPs are related to the stretching vibrations of C=O and C-N carbonyl groups associated with the formation of an amide bond between carboxylic acid, ursolic acid, and chitosan amine [22]. The peak at 1550 cm–1 is attributed to the N-H bending vibrations and the wide peak around 3400 cm–1 is related to the O-H stretching vibrations at the Ch surface. The emergence of these peaks in the spectrum of UA-Ch-NPs and the absence of the characteristic peaks of UA confirm the binding interaction between Ch and UA through the formation of amide bonds.The TEM image of the synthesized UA-Ch-NPs is shown in Figure 1a. As shown, spherical nanoparticles with an almost uniform particle size of 75 nm were obtained. The FTIR spectra of UA, Ch, and UA-Ch-NPs are presented in Figure 1b. A unique characteristic peak in the FTIR spectrum of UA at 3520 cmThe results of DLS and zeta potential analysis of UA-Ch-NPs are presented in Figure 1c. Accordingly, the average particle size of UA-Ch-NPs is 258 nm, and its polydispersity index (PDI) is 0.409. Based on the results, the zeta potential of UA-Ch-NPs is +40.1 mV, which is associated with positively charged amine groups on the Ch surface. The XRD patterns of UA and UA-Ch-NPs are shown in Figure 1d. The characteristics peaks associated with UA were observed at 2θ values of 5.15, 8.81, 10.30, 14.50, 16.43, 21.40, and 27.22. These characteristic peaks were also observed in the diffraction pattern of the UA-Ch-NPs indicating the loading of UA on Ch nanoparticles. Ch usually shows a broad diffraction peak at 2θ = 22.6 ° which is also seen in the UA-Ch-NPs diffraction along with some peaks related to UA. The presence of similar characteristic peaks related to UA and UA-Ch-NPs was reported in a previous research [24].S. aureus strains, isolated from bovine mastitis, showed different levels of antimicrobial susceptibility patterns. The antimicrobial resistance (AMR) of S. aureus isolates is shown in Figure 2a. Generally, the high resistance or low susceptibility to antibiotics was observed with penicillin and ciprofloxacin where 66.2% and 76.2% of the isolates were resistant, respectively. Gentamicin revealed the most effective antibiotic effect, and all the S. aureus strains were sensitive to this antibiotic (100% sensitive). The MIC value for UA and UA-Ch-NPs were 64 and 32 µg/mL, respectively , revealing that the growth-inhibitory effects of UA-Ch-NPs were more than those of UA. Results also showed that S. aureus was sensitive to UA and UA-Ch-NPs at MIC, 0.5 MIC, and 2 MIC concentrations . Antibiotic sensitivity of 21  S. aureus, 21 strains isolated from bovine mastitis were cultured. As determined by microtiter plate assay (OD640), among the 21 isolates, biofilm-producing bacteria were classified as moderate (5 isolates) and strong (16 isolates). After 24 h of incubation of the bacteria with UA and UA-Ch-NPs, biofilm formation reduced drastically . A number of studies have indicated a potential effect of bacterial hydrophobicity on bacterial adhesion to host tissues and biofilm formation. Therefore, cell surface hydrophobicity (CSH) and biofilm formation are significantly correlated . Three types of hydrophobicity were identified: weak , moderate (> 30%–70%), and strong (> 70%). In this study, among the 21 isolates of bacterial strains, 62% had strong CSH, and the rest exhibited moderate hydrophobicity. As summarized in Table, among the S. aureus isolates, most strains were strongly hydrophobic. To further study the antibiofilm characteristics of UA-Ch-NPs, AFM analysis was used for the investigation of the interaction between the bacterial cells and the nanoparticles. AFM images of S. aureus showed a significant difference between biofilm formation of the control sample (S. aureus without treatment) and UA-Ch-NPs treatment . Several research studies showed that the icaD and icaA genes of S. aureus have a key role in the synthesis of polysaccharide intercellular adhesion from poly-N-acetylglucosamine contributing to biofilm formation [26]. Our data also showed that UA-Ch-NPs led to a dramatic reduction in both icaA and icaD genes . The statistical analysis showed that the results were statistically significant (p < 0.05).To determine the effects of UA and UA-Ch-NPs on biofilm formation byIn the present study, UA-Ch-NPs were synthesized, and their antiinfective properties were investigated. Several analytical techniques were used to characterize the developed UA-containing nanoparticles to evaluate their suitability for the intended purpose. The TEM analysis showed that the synthesized UA-Ch-NPs had a good monodispersity with spherical morphology. The XRD analysis results confirmed the presence of UA on the Ch nanoparticles and from the FTIR results, the successful interaction of UA with the Ch nanoparticles surface was deduced. The DLS analysis of UA-Ch-NPs showed an average particle size of around 258 nm with a PDI of 0.409. The PDI is a value between 0 and 1. The closer this value is to zero, the greater is the uniformity of the particle size. The best value is around 0 to 0.2 for polymeric nanoparticles; for drug release purposes, this index should be below 0.4, and for other purposes, up to 0.7 is acceptable and shows the uniformity of the particle size [27]. Accordingly, the developed nanoparticles with a PDI of around 0.41 were suitable for the drug delivery purpose. The hydrodynamic diameter obtained from the DLS analysis was 258 nm, which was different from that obtained from the TEM analysis in a dry state (vacuum). This difference in particle size between DLS (258 nm) and TEM (75 nm) is attributed to the positive zeta potential of these nanoparticles that are strongly covered by the water molecules while being dispersed in aqueous solution for DLS analysis and show a large hydrodynamic diameter compared to that obtained from TEM analysis in dry state. The obtained diameter for the synthesized UA-Ch-NPs in this study is in the middle range compared to the other studies on UA-loaded nanocarriers. For instance, an average particle size of 330 nm was reported for UA-loaded Ch/poly (lactic acid) (PLA) nanoparticles [28], while another study reported a particle size of around 100–200 nm (an average of 120 nm) for UA-loaded Ch nanoparticles [10]. There is not a consensus among researchers about the suitable particle size to be absorbed by the intestinal mucosa. Some investigations have reported that particles with the size 130–950 nm are suitable to be absorbed by intestinal M cells [29]. The oversized pores of tumor microvessels varies from 100–1200 nm [30], hence, nanoparticles with a size of about 250 nm size could enter the broad fenestrations of the tumor tissues, while they are not able to enter the vessels of the normal endothelium which are narrow, approximately 5–10 nm [23]. Hence, the particle size obtained in this study has a relatively suitable size for drug delivery applications.The zeta potential of the prepared nanoparticles was +40.1 mV demonstrating their high stability and the positive charge of the particles, which provide an effective system for drug delivery due to being taken up by proliferating cells. This effect was confirmed by previous studies [31]. The binding of nanoparticles to the cell membrane is affected by the charge of the NPs. Since cellular surfaces are dominated by negatively charged sulphated proteoglycans molecules that are highly anionic, the interaction between the proteoglycan and the nanoparticle shell (positively charged) is ionic, so the higher surface charge of the nanoparticles results in a stronger attachment of the nanoparticles to the cell membrane and higher adsorption of the nanoparticles [32]. Antonio et al. [28] also reported that by the incorporation of Ch to the surface of UA-loaded PLA nanoparticles, the zeta potential increased from –25 to +28 mV because of the amino groups of the Ch molecules, which facilitated the interaction of the nanoparticles with biological membranes.S. aureus and its biofilm formation ability were observed. While S. aureus was resistant to antibiotics, it showed high susceptibility to UA and UA-Ch-NPs. In a study by Wu et al. [33], the stable antibacterial effect of triterpene derivatives such as UA was also demonstrated. In addition, the reduction of the MIC value for UA-Ch-NPs compared to UA is an indication of the higher growth-inhibitory effects of UA-Ch-NPs, which is confidently associated with the presence of Ch. It has been reported that Ch derivatives can interact with microbial cell membranes and ultimately decrease membrane integrity [34]. Qi et al. [35] also reported that Ch-NPs act against S. aureus by inhibiting their growth through membrane disruption and leakage of metabolites.In vitro studies demonstrated the antiinfection activity induced by UA-Ch-NPs. The antimicrobial effects of UA-Ch-NPs on the growth of S. aureus infection. Our findings also indicated that UA-Ch-NPs were able to inhibit bacterial growth in a concentration-dependent manner. According to Figure 2c, the developed UA-Ch-NPs prolonged the lag phase of S. aureus and also reduced the OD. Generally, during the lag phase, cells are depleted of metabolites and enzymes because they are adapting to their new growth conditions. In this phase, intermediates, enzymes, RNA, and other molecules are made to resume the growth. In this situation, the occurrence of a long lag phase is probable, which represents the period required for sufficient multiplication of a few mutants in the inoculum for an apparent rise in cell number [36]. This study mainly aimed to produce a nanoformulation to combat S. aureus isolates. Emerging data is trying to characterize genes engaged in biofilm formation. For example, the icaA and icaD genes have been extremely characterized before being engaged in S. aureus biofilm formation [36]. These genes contribute to the intercellular attachment of bacteria and biofilm formation. Our data also clearly indicated that the treatment of bacterial cells with UA-Ch-NPs significantly reduced icaA and icaD, and consequently, biofilm formation. Qian et al. [37] also reported that UA disrupted the cell membrane integrity of carbapenem-resistant Klebsiella pneumoniae, and strong inhibitory influences against the expression of biofilm-related genes and biofilm formation were reported.In addition, our findings also showed that UA-Ch-NPs reduced the biofilm formation by the S. aureus revealed the enhanced inhibitory effect of the UA-loaded nanoparticles due to the presence of Ch nanoparticles. This pharmaceutical agent reduced the growth of S. aureus and its biofilm formation, which was also confirmed by the AFM analysis. The treatment of bacterial cells with UA-Ch-NPs significantly decreased the expression of icaA and icaD genes which are engaged in biofilm formation. The results of this study indicated that UA-Ch-NPs could serve as an antibiotic agent, however, this needs to be confirmed by further research in the future. The current study managed to produce UA-Ch-NPs successfully. According to the TEM analysis, the synthesized nanoparticles were uniform in size with a good monodispersity. Moreover, the FTIR analysis results showed the interaction between functional groups of UA and Ch and thereby the successful loading of UA on the nanoparticles. The XRD results also showed the characteristic peaks associated with UA in UA-Ch-NPs. According to the results, a decrease in the MIC value of UA-Ch-NPs in comparison with UA against


The mitogen-activated protein kinase (MAPK) p38 is an essential family of kinases, regulating responses to environmental stress and inflammation. There is an ever-increasing plethora of physiological and pathophysiological conditions attributed to p38 activity, ranging from cell division and embryonic development to the control of a multitude of diseases including retinal, cardiovascular, and neurodegenerative diseases, diabetes, and cancer. Despite the decades of intense investigation, a viable therapeutic approach to disrupt p38 signaling remains elusive. A growing body of evidence supports the pathological significance of an understudied atypical p38 signaling pathway. Atypical p38 signaling is driven by a direct interaction between the adaptor protein TAB1 and p38α, driving p38 autophosphorylation independent from the classical MKK3 and MKK6 pathways. Unlike the classical MKK3/6 signaling pathway, atypical signaling is selective for just p38α, and at present has only been characterized during pathophysiological stimulation. Recent studies have linked atypical signaling to dermal and vascular inflammation, myocardial ischemia, cancer metastasis, diabetes, complications during pregnancy, and bacterial and viral infections. Additional studies are required to fully understand how, when, where, and why atypical p38 signaling is induced. Furthermore, the development of selective TAB1-p38 inhibitors represents an exciting new opportunity to selectively inhibit pathological p38 signaling in a wide array of diseases. The p38 mitogen-activated protein kinase (MAPK) family are critical cellular signaling regulators that drive many physiological and pathophysiological pathways. Therefore, it is not surprising that since their discovery in 1994 . As suchIn light of the sheer volume of p38 research articles and the wealth of excellent reviews available, it would be impractical and redundant to cover all aspects of p38 MAPK signaling. Therefore, this review will initially provide a brief overview of the history of p38 and the many roles it plays in disease progression. This will be followed by a more focused examination of the novel atypical p38 activation pathways, specifically including atypical p38 activation by GPCRs, their implications for disease progression, and therapeutic intervention. In comparison to classical p38 activity, atypical p38 signaling has been understudied with only 44 publications, however, this growing body of work represents a fresh perspective on p38 activity and function in disease.The classical pathway for MAPK activation is through a three-tiered kinase cascade, where MAP kinase kinase kinases activate a MAP kinase kinase which in turn activate MAPKs such as p38 . The mos−/−mkk3, −/−mkk6), suggesting functional conservation [MKK6 and MKK3 share a high degree of sequence homology with an 86% amino acid identity and selectively activate p38 MAPK over other MAP2Ks ,9. MKK3/ervation , while rervation . MAP2Ks are activated by MAP3Ks, which are less specific than MAP2Ks and activate an array of regulatory proteins. MAP3Ks are categorized into three broad families: MAPK/ERK kinase (MEK) kinases, mixed lineage kinases (MLKs), and thousand and one kinases (TAOs) . Over 50There are four isoforms of p38 . MAPK p38α is the founding member of the family and is ubiquitously expressed throughout the body. The four isoforms share a high degree of homology, p38β with 74% homology to p38α, p38γ with 63% homology to p38α, and p38δ with 60% homology to p38α ,22,23. CNotably, p38α is the only isoform that is essential for embryonic development, where it regulates placental vasculogenesis and morphogenesis ,26. AddiThe differential activation and signal transduction by MAPKs appear to be in part regulated by binding to specific scaffold proteins ,30,31,32As the downstream signal transduction pathways for p38 are highly complex, we refer the reader to several outstanding and exhaustive reviews ,36,37. BThere are over 100 substrates identified for the p38 family with selective activation of specific substrates determined by the stimulation mechanism, including inflammation, DNA repair, cell differentiation, stem cell physiology, stress responses, and neuronal function ,36,44. AWith p38 MAPK playing a critical role in many cellular functions, dephosphorylation of both threonine and tyrosine residues in the active loop is required for inactivation of the kinase and signal termination. The most widely studied family responsible for dephosphorylating p38 is the dual specificity phosphatases (DUSPs) also referred to as MAPK phosphatases (MKPs). The DUSP family can dephosphorylate all members of the MAPK family. However, DUSP1/MKP1, DUSP10/MKP5, DUSP26/MKP8, and DUSP12 display a higher degree of specificity to p38α than the other DUSP family members ,46,47, wSince its discovery, p38 has been recognized as a potentially critical therapeutic target ,53. MultThe consistent short-lived efficacy of current inhibitors suggests that compensatory inflammatory pathways are upregulated over time in response to total p38 activity inhibition. While many well-designed investigations have studied p38 as a therapeutic target, Much remains unknown about p38 subcellular localization and what controls its access to downstream substrates after stimulation, especially pertaining to MKK3/6 verses atypical activation. Current investigations into inhibitor design are shifting away from targeting the catalytic site of p38 and instead focus on substrates and downstream signaling pathways ,69,70,71The burgeoning generation of selective atypical targets provides a promising new direction for clinically viable approaches for anti-p38 therapeutics. Furthermore, it is predicted that the combinatory therapies described above will provide a template moving forward to enable clinically viable strategies to target p38 activity.MKK3/6 kinase activity is widely considered to be the primary mechanism for p38 phosphorylation. Nevertheless, there is a growing body of evidence to support alternative mechanisms for p38 activation . Two “at48-linked ubiquitination to regulate TAB1 turnover/degradation. TAB1 expression is significantly elevated in the absence of itch, leading to enhanced atypical p38 activation and increased cytokine production, including interleukin-6 (IL-6), interleukin-1beta (IL-1β), interleukin-11 (IL-11), and interleukin-19 (IL-19). Critically, Wang et al. in 2013 developed a peptide inhibitor fused to the HIV-TAT peptide, generating a cell-penetrating peptide inhibitor that selectively disrupts the TAB1 interaction with p38, substantially attenuating atypical p38 activation [−/− mice, the peptide blocked atypical p38α signaling and dermal inflammation was significantly suppressed [Reactive oxygen species are thought to be the initial driving force behind atypical p38 signaling in cardiac ischemia-reperfusion damage ,72. Simitivation ,84. Whenppressed . Furtherppressed ,72,85, appressed ,72. Critppressed ,86, the ppressed .This critical interaction provides a novel opportunity to further develop the peptide inhibitors or screen for small molecule inhibitors to target atypical p38 signaling selectively. Indeed, using a virtual small fragment screen, a group of functionalized adamantanes, specifically 3-amino-1-adamantanol, was found to bind to a critical hydrophobic pocket, forming hydrogen bonds with two key residues, leucine 222 and 234, in the non-canonical TAB1 binding site on p38α. Further screening found there to be three distinct fragment binding sites within the non-canonical binding site. Linking sulfonamide scaffolds to the adamantanol generated a small molecule with a high affinity to the three regions in the non-canonical binding site . AdditioDespite these detailed studies describing the exact molecular mechanism of TAB1-p38α interaction and degradation, there are significant gaps in our understanding, specifically for how osmotic stress, oxidative stress, LPS, or inflammatory cytokines such as TNF-α and IL-1β initiate the TAB1-p38α interaction and atypical p38 signal transduction. Conversely, recent studies have shown that a family of G protein-coupled receptors (GPCRs) can initiate the TAB1-p38 interaction through a novel ubiquitin-driven pathway . This pathway is critical for T-cell activation through a T-cell receptor (TCR) specific mechanism . In contq or Gα12/13 to induce activation of the proto-oncogene tyrosine-protein kinase c-Src (Src short for sarcoma) and subsequent activation of the E3 ubiquitin ligase, neural precursor cell expressed developmentally downregulated 4-2 (NEDD4-2) [As the most extensive and versatile family of membrane proteins, G protein-coupled receptors (GPCRs) regulate many cellular pathways by activating MAPKs via G protein-dependent and -independent mechanisms ,92,93,94NEDD4-2) . NEDD4-2NEDD4-2) ,87. C-SrNEDD4-2) , althougNEDD4-2) ,95. HoweNEDD4-2) ,32,87,95NEDD4-2) ,100. IntNEDD4-2) . It is sImportantly, this pathway is not unique just to PAR1 and α-thrombin. NEDD4-2 dependent regulation of atypical p38 signaling is also conserved for the purinergic receptor P2Y1. Furthermore, a recent study also demonstrated that the pathway is conserved for prostaglandin E2 (PGE2), histamine, ADP, and α-thrombin-mediated p38 activation and inflammatory cytokine production in primary human microvascular and macrovascular endothelial cells . AdditioAs p38 MAPKs play a critical role in the modulation of many physiological processes, the dysregulation of their signaling pathways can result in the pathogenesis of a range of inflammatory diseases, neurological diseases, retinopathies, and cancers. There have been multiple recent outstanding studies and reviews that extensively cover the many pathological pathways controlled by classical p38 signaling, some examples are highlighted in Early studies revealed that p38 MAPKs have a central role in the development of various chronic inflammatory diseases due to pro-inflammatory cytokine (PIC) production ,164. SpeLikewise, there is strong evidence for p38 in both tumor suppressive cellular homeostasis, balancing proliferation, differentiation, and apoptosis, and tumor promoting roles through promoting cell survival, proliferation, and angiogenesis . FurtherIn a similar manner, MAPK p38-induced cytokine expression during neuroinflammation accelerates the development of chronic neurodegenerative diseases such as multiple sclerosis (MS) , AlzheimContrary to the highly studied MKK3/6-dependent pathway, the impact of TAB1-p38-dependent signaling in physiology and disease remains largely understudied with just 44 research articles on the subject . As ment−/− ischemic mouse, the TAB1-p38 interaction was a leading contributor to necrosis in cardiomyocytes [There is a growing awareness that atypical p38α activation plays a key role multiple p38 driven pathologies. The initial studies describing atypical p38α activation demonstrate its role in ischemic cardiac damage, ischemia-reperfusion injury, and amyloidosis. In an MKK3myocytes . The rolmyocytes . Supportmyocytes , and cycmyocytes . Additiomyocytes . Inhibitmyocytes . Additiomyocytes ,185.−/− mice, TAB1 expression is significantly enhanced, leading to robust p38 autophosphorylation and subsequent increases in inflammatory cytokine expression, immune cell recruitment, and spontaneous skin lesions [Aside from the heart, p38 autophosphorylation has also been indicated in pathological inflammation in dermal disorders, preterm birth, and more broadly in vascular inflammation. In the itch lesions . The use lesions . In the  lesions . Atypica lesions .Vascular inflammation also directly activates GPCR-dependent p38 signaling in endothelial cells. In these studies, GPCR ligand α-thrombin induces endothelial barrier disruption driving vascular leakage and permeability. Additionally, recent studies of GPCR-mediated TAB1-p38 activity have demonstrated that it is conserved in multiple endothelial vascular beds and activated by a family of GPCR ligands associated with inflammation such as histamine, PGE2, ADP, and potentially many others ,32,87. W+ T-cells through increased expression of TAB1 following antigen exposure and abrogating TCR [Toxoplasma gondii, resulting in pro-inflammatory IL-12 production specific to atypical signaling [Leishmania infection results in parasite GP63-induced degradation of TAB1 to reduce p38 activation [Beyond the vasculature, the role of atypical p38 is also explored in the modulation of the immune system by inflammatory ligands, attenuation of the TCR, and response to pathogens. Basophils and eosinophils isolated from healthy patients undergo p38 autophosphorylation in response to cytokine exposure from TNFα and GM-CSF, contributing to prolonged inflammation like that seen in pulmonary inflammatory disorders . Conversting TCR . Similarting TCR . AMPK alting TCR . Intraceignaling . Leishmativation , the revtivation . These sAnother example of atypical p38 activation comes from a recent study that demonstrated that multiple viruses utilize atypical p38 signaling to drive viral infections. Inhibition of TAB1-dependent p38 activation impaired hepatitis C virus (HCV) assembly and viral replication. This was also confirmed for severe fever with thrombocytopenia syndrome virus (SFTSV), herpes simplex virus type 1 (HSV-1), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) . Indeed,In the realm of type 1 diabetes, a link was found for TAB1-p38 interaction in the apoptosis of beta cells via oxidative stress by NO  and cytoH1 and TH17) and expression of IFNγ and IL17. Indicating that both the classical and atypical p38 activation pathways could work synergistically to induce a balance between pro- and anti-inflammatory responses [Contrary to TAB1-dependent signaling, Zap70-dependent activation of p38 is exclusive to T-cell activation via the TCR response, which is negatively regulated by p38 phosphorylation of upstream Zap70 ,89. Howeesponses . It is cesponses .The 25-year history of p38 MAPK has clearly demonstrated that this family of inflammatory kinases are essential for normal physiological processes and, if dysregulated, can be significant contributors to many diseases. Yet, despite many outstanding studies and carefully controlled clinical studies, therapeutic interventions targeting the conserved ATP pocket or structural scaffolds have so far been unsuccessful in the clinic. However, there are some promising avenues like targeting downstream signaling transducers such as MK2. Furthermore, the selective inhibition of pathological atypical p38 signaling represents a significantly under-investigated avenue and potentially critical target for therapeutic intervention.Although there has been important progress in understanding the structural basis of the TAB1-p38 interaction and a clear mechanism has been defined for GPCR induced activation of atypical p38 signaling, there remain many gaps in our understanding of where, when, and why this pathway exists. There is still little understanding of how atypical p38 signaling alters the functional outcome of p38 activation to drive disease progression.As outlined above, there is a growing body of clear evidence describing TAB1-dependent atypical p38 signaling . AtypicaBased on the significant role of GPCR ligands and p38 in the progression of so many diseases, it is clear that the current research has only just scratched the surface of the potential import of atypical p38 signaling. Future studies will yield critical detail to the broader mechanism of activation, and the development of TAB1-p38-selective inhibitors could pave the way forward to developing a clinically viable therapeutic.


SOCS1 and USP9X following ZIKV infection to regulate Vav protein degradation or activation, respectively, and impact Vav/Rac/RhoA signaling in NPC and neurons. Experiments to define these interactions will further our understanding of the molecular basis of CZS and potentially other developmental disorders stemming from in utero infections. Additionally, Vav/Rac/RhoA signaling pathways may present tractable targets for therapeutic intervention or molecular rationale for disease severity in CZS.Zika virus (ZIKV) infection during pregnancy can result in a significant impact on the brain and eye of the developing fetus, termed congenital zika syndrome (CZS). At a morphological level, the main serious presentations of CZS are microcephaly and retinal scarring. At a cellular level, many cell types of the brain may be involved, but primarily neuronal progenitor cells (NPC) and developing neurons. Vav proteins have guanine exchange activity in converting GDP to GTP on proteins such as Rac1, Cdc42 and RhoA to stimulate intracellular signaling pathways. These signaling pathways are known to play important roles in maintaining the polarity and self-renewal of NPC pools by coordinating the formation of adherens junctions with cytoskeletal rearrangements. In developing neurons, these same pathways are adopted to control the formation and growth of neurites and mediate axonal guidance and targeting in the brain and retina. This review describes the role of Vavs in these processes and highlights the points of potential ZIKV interaction, such as (i) the binding and entry of ZIKV in cells via TAM receptors, which may activate Vav/Rac/RhoA signaling; (ii) the functional convergence of ZIKV NS2A with Vav in modulating adherens junctions; (iii) ZIKV NS4A/4B protein effects on PI3K/AKT in a regulatory loop via PPI3 to influence Vav/Rac1 signaling in neurite outgrowth; and (iv) the induction of  Aedes spp. mosquitoes, ZIKV can also be transmitted between humans through blood, sexual contact and vertically [Zika virus (ZIKV) is a positive-sense single-stranded RNA virus of the flavivirus genus, which includes other viruses such as dengue virus, West Nile virus, Japanese encephalitis virus and yellow fever virus ,2. Althortically ,8.Approximately 80% of adult ZIKV infections are asymptomatic, but in those adults who are symptomatic, the clinical manifestations include fever, rash, myalgias/arthralgias, malaise and headaches, lasting for up to 7 days ,8,9. ZIKMaternal ZIKV infection in any of the three trimesters of pregnancy can result in vertical transmission of ZIKV to the developing fetus where adverse outcomes can ensue . This isThe underlying pathogenic mechanisms of CZS at a cellular level remain incompletely understood. There is a suggestion that the pathogenesis of CZS may in-part involve inflammatory changes and dysregulation of interferons in the developing brain ,21. MicrVavs are a family of proteins that function as guanine exchange factors (GEF) that activate Rho family GTPases, such as RhoA, Rac1 and Cdc42 ,30,31,32VAV2 in particular displays a strong developmental transcript expression pattern across all regions of the human brain, with high levels of expression in utero, which is dramatically downregulated postnatally, suggesting a fundamental role in early brain development (Vav2 mRNA with time in the hippocampus throughout in utero development (36), the presence of Vav2 protein in early postnatal retinal ganglion cells, embryonic cortical and hippocampal neurons and a decrease in Vav2 protein with time in brain lysates throughout embryonic and early postnatal development in the mouse (51).There are three members of the Vav family of proteins in mammals—Vav1, Vav2 and Vav3—which exhibit similar but not identical functions, as reviewed elsewhere ,32,39,40elopment . This isAt a molecular level, Vavs function intracellularly in cellular signaling pathways, downstream of receptors with either intrinsic or associated tyrosine kinase activity ,40 Figu. These rThe development of the brain is a complicated and tightly regulated process ,69. The Cdc42, leads to a progressive loss of adherens junctions and apico-basal polarity and is associated with differentiation and exit of VZ NPCs [Rac1 knockout results in disrupted VZ architecture leading to premature NPC differentiation associated with increased cell cycle exit and reduced NPCs proliferation and self-renewal [Rac1 from the VZ of the telencephalon recapitulated this phenotype, and further demonstrated increased apoptosis [RhoA deletion in the spinal cord of embryonic mice resulted in reduced NPC pools via reduced proliferation and increased cell-cycle exit [RhoA knockout in the midbrain and forebrain of mice also resulted in a disruption to NPC adhesion and polarity of VZ NPCs [As mentioned above, NPCs maintained within the VZ are self-renewing and there is extensive literature linking the Rho signaling pathway to the polarity, adhesion and VZ localisation of NPCs and therefore their self-renewal ,35,82. C VZ NPCs . The out VZ NPCs . Likewis-renewal . Conditi VZ NPCs . Intrigu VZ NPCs . Both th VZ NPCs ,82. Coll VZ NPCs ,84,85. AVav3 knockout exhibited an accelerated differentiation of retinal NPCs into cone and retinal ganglion cells, and more cells exhibiting markers of late retinal progenitor cells [Evidence for this mechanism comes from studies in the developing eye . Live ceor cells . This suXenopus spinal neurons, where Vav2 overexpression inhibits RhoA signaling and promotes neurite growth [Following their production during neurogenesis, neurons must mature to form functional circuitry within the brain. A major event in maturation is neuritogenesis, the growth and arborization of neurites including dendrites and axons that enable them to project from the neuronal cell body and connect with other neurons throughout the brain. A key structure driving this process is the growth cone, located at the leading edge of outgrowing neurites . The deve growth .Vav2 and Vav3 knockout mice exhibit fewer ipsilateral retinogeniculate axonal projections and demonstrate skewing of axon positioning in the thalamus [Vav3−/− mouse brains revealed reduced expression of GABAergic axon terminal markers in the ventrolateral medulla (VLM), while axonal tracing revealed a failure of caudal VLM GABAergic axonal projections to properly innervate their targets in rostral aspects of the VLM [By mediating neurite growth cone growth or collapse, Vav also regulate signals that appropriately guide and target axons as they navigate through the brain. The cell–cell signaling between Eph and Ephrin receptors is a well-established axon guidance mechanism and involves activation of Vav ,99,100. thalamus . Severalthalamus . Immunoh the VLM . Further the VLM . In dros the VLM ,102.Collectively, these data implicate Vav signaling pathways in a range of processes for neuronal cell maturation including neurite outgrowth and axon targeting in a variety of developing CNS tissues including the eye. The major site of Vav activity is in the growth cone, and the mechanisms of Vav action typically converges to the regulation of Rho, Rac1 and Cdc42. These same neuronal maturation processes are disturbed in CZS, and this places Vavs as candidate mediators of CZS pathogenesis.Vav may be linked to the pathogenesis of CZS through the process of viral entry into cells of the CNS. The TAM receptors although not obligatory for infection, can mediate ZIKV entry into cells including NPC, microglia and astrocytes ,104,105.ZIKV-NS2A expression in developing mouse cortices and ZIKV infection of developing brains reduces proliferation and induces premature differentiation of RGCs, as well as aberrant neuronal positioning and cortical arrangement . SpecifiOther ZIKV proteins have been implicated in disrupting neural stem cell development and autophagy through the PI3K/AKT pathway . SpecifiSOCS1 and 3 and a reduction of type I and III IFN responses [USP9X is increased by ZIKV infection [A normal and important response to viral infection is the induction of interferon (IFN) and inflammatory pathways, with the JAK/STAT signaling pathway key to many of these immune/inflammatory responses. This pathway can be influenced by Rho GTPases, such as Rac1 in response to IFN-γ  and the esponses . SOCS1 hesponses . Vav alsesponses ,121, whiesponses . USP9X hesponses ,124. Thenfection  and thusVav proteins have been shown to be key players in multiple cellular and physiological processes, including neurodevelopment. In particular, Vav/Rho GTPase signaling pathways influence NPC function including their cell adhesion, polarity, proliferation, differentiation fate and survival. Additionally, Vav/Rho GTPase signaling affects neuronal cell functions including neurite growth cone regulation, axon projection and targeting. This presents a circumstantial association of Vav/Rho GTPase signaling pathways with the pathogenesis of CZS—where these aspects of neurodevelopment are also disrupted. Potentially, ZIKV may influence Vav/Rho GTPase signaling by stimulating TAM-Vav-dependent signaling pathways following ZIKV entry; ZIKV alterations in Vav-regulated adherens junction formation; ZIKV inhibition of Vav through inhibition of PI3K/AKT pathways and reduced production of PIP3; or via SOCS1-mediated ubiquitination and degradation, or USP9X-mediated activation of Vav proteins.Further investigation of these interactions is warranted to define the molecular pathways driving the pathogenesis of CZS and potential therapeutic targets to prevent NPC and neuronal cell dysfunction. These outcomes may also have an impact on understating of infection-driven neurodevelopmental pathologies that impact the NPC pool . AdditioVAVs that can influence infectious diseases, such as susceptibility to Candida albicans [VAV polymorphisms in relation to CZS could lead to useful clinical management strategies or prognostic information.Additionally, there are known polymorphisms in albicans . Given t


The global prevalence of metabolic diseases, such as obesity, diabetes, and atherosclerosis, is rapidly increasing and has now reached epidemic proportions. Chronic tissue inflammation is a characteristic of these metabolic diseases, indicating that immune responses are closely involved in the pathogenesis of metabolic disorders. However, the regulatory mechanisms underlying immunometabolic crosstalk in these diseases are not completely understood. Recent studies have revealed the multifaceted functions of semaphorins, originally identified as axon guidance molecules, in regulating tissue inflammation and metabolic disorders, thereby highlighting the functional coupling between semaphorin signaling and immunometabolism. In this review, we explore how semaphorin signaling transcends beyond merely guiding axons to controlling immune responses and metabolic diseases. The prevalence of metabolic diseases, such as obesity, diabetes, and atherosclerosis, is rapidly increasing, despite extensive research into their pathogenesis. Although chronic tissue inflammation indicates the involvement of immune responses in the pathogenesis of metabolic disorders, the regulatory mechanisms of immunometabolic crosstalk in these diseases are not fully understood.Semaphorins are a large family of secreted and membrane-associated molecules that are essential for the development of the nervous system. The semaphorin family, which is characterized by an extracellular N-terminal Sema domain and plexin–semaphorin–integrin domains, is composed of eight subclasses . ClassesAmong the semaphorin subclasses, class III semaphorins are the most widely studied molecules in the regulation of obesity and systemic metabolism Fig. . In 2013Sema3G, another class III semaphorin, plays key roles in adipogenesis . Sema3G In addition to their functions in adipose tissues, class III semaphorins regulate systemic energy balance by modulating hypothalamic neural circuits. Klaauw et al.  reportedMecp2 deficiency results in spontaneous obesity due to decreased sympathetic innervation in BAT. Mecp2−/− macrophages overexpress Plexin-A4, which suppresses the axonal outgrowth of Sema6A-expressing sympathetic nerves and innervation in BAT. Although the mechanism by which MeCP2 suppresses Plexin-A4 expression in macrophages remains unclear, this study revealed the essential roles of interactions between macrophages and neurons via the Sema6A–Plexin-A4 signaling axis in the regulation of metabolic homeostasis.With respect to the other semaphorin classes, the Sema6A–Plexin-A4 signaling axis regulates homeostatic energy expenditure and thermogenesis of brown adipose tissue (BAT) by modulating sympathetic innervation. Wolf et al.  revealedAccumulating evidence has elucidated the mechanisms underlying the pathogenesis of diabetes, leading to the clinical application of various drugs, such as metformin, sulfonylureas, glinides, thiazolidinediones, dipeptidyl peptidase-4 inhibitors, glucagon-like peptide-1 receptor agonists, sodium-dependent glucose cotransporter 2 inhibitors, and insulin . HoweverDiabetic retinopathy (DR) is a common complication associated with diabetic macular edema, vitreous hemorrhage, traction retinal detachment, and macular ischemia . AlthougIn contrast, the Sema3E–Plexin-D1 signaling axis normalizes angiogenic directionality in both developing retinas and ischemic retinopathy . Neuron-Sema4d−/− mice show attenuated pathological retinal neovascularization and vascular leakage in a streptozotocin-induced diabetes or oxygen-induced retinopathy model, indicating the pathological roles of Sema4D signaling in retinopathy. Sema4D–Plexin-B1 signaling impairs endothelial cell function through mDIA1, which functions as an adaptor protein of Src kinase. In addition, Sema4D–Plexin-B1 signaling induces pericyte migration and N-cadherin internalization, which exacerbates vascular permeability. Moreover, anti-Sema4D and anti-VEGF antibodies have synergistic effects in inhibiting retinal neovascularization and vascular leakage, indicating the promising potential of Sema4D as a therapeutic target for retinopathy.In addition to class III semaphorins, the Sema4D–Plexin-B1 signaling axis contributes to the development of diabetic retinopathy . DR patiDiabetic nephropathy (DN) is the leading cause of hemodialysis worldwide. Hyperglycemia, the primary pathogenic factor responsible for the development of DN, causes hypertension, altered tubuloglomerular feedback, renal hypoxia, and podocyte injury, leading to progressive glomerular sclerosis and impaired glomerular filtration . Sema3A–Sema3g−/− mice exhibit only partially aberrant podocyte foot process structures without significant glomerular defects. However, Sema3g−/− mice exhibit increased albuminuria in a lipopolysaccharide (LPS)-induced acute inflammation model or a streptozotocin-induced diabetes model. Moreover, the administration of LPS in mice with podocyte-specific deletion of Sema3G results in enhanced expression of inflammatory cytokines, such as chemokine ligand 2 and interleukin 6. Overall, podocyte-derived Sema3G is protective against kidney inflammation.In contrast to the pathogenic roles of Sema3A in DN, Sema3G protects podocytes from inflammatory kidney diseases and DN . AlthougDiabetes perturbs bone remodeling and induces osteoporosis . SemaphoSema6a mRNA. miR-27b augments bone marrow-derived angiogenic cell function in diabetes by suppressing Sema6A expression, leading to accelerated wound healing. These studies highlight the close association between semaphorin signaling and diabetic wound healing. Due to the suppression of Sema3C–Nrp-2 signaling, diabetes induces corneal dysfunction, such as delayed epithelial wound healing and nerve regeneration [Delayed wound healing is a major complication of diabetes. Delayed and impaired healing causes diabetic foot ulcers, which can result in lower limb amputation . Sema4D neration . AlthougDisrupted lipid metabolism causes atherosclerosis, a chronic systemic inflammatory disease characterized by the accumulation of lipids and inflammatory immune cells in the intima of blood vessels. Recent studies have revealed the essential role of semaphorin signaling in the development of atherosclerosis  Fig. 3)3).Fig. Sema4d reduces dyslipidemia-induced platelet hyperactivity and ameliorates atherosclerosis in low-density lipoprotein receptor (LDLR)-deficient mice fed with a high-fat diet. Our previous study also showed that loss of Sema4d in apolipoprotein-E (ApoE)-deficient mice retards the progression of atherosclerosis [The association between semaphorin signaling and atherosclerosis was first identified in 2009 . Given tclerosis . Sema4D,Sema3A exerts atheroprotective effects by inhibiting leukocyte rolling, adhesion, and transmigration into the subendothelial wall during atherogenesis . In LDLRNos2 and Arg1, respectively. Nos2 and Arg1 are marker genes of inflammatory and anti-inflammatory macrophages, respectively. Sema3E binding to Plexin-D1 inhibits the directional migration of macrophages by disrupting the Rho GTPase signaling cascade, actin cytoskeleton reorganization, and polarization. In contrast, the Sema3E–Plexin-D1 signaling axis inhibits the proliferation and migration of vascular smooth muscle cells (VSMCs), which are hallmarks of atherosclerosis and restenosis [The Sema3E–Plexin-D1 signaling axis promotes the development of atherosclerosis via the retention of inflammatory macrophages in the plaque . In ApoEstenosis . In a moApoe−/− mice, endothelial-specific Plexin-D1 knockout (Plxnd1iECKO) mice fed a high-fat diet exhibit a significant decrease in the plaque burden of both the whole aorta and the aortic arch. In contrast, Plxnd1iECKOApoe−/− mice show an increase in plaque burden in the descending aorta. Thus, the endothelial Plexin-D1–Nrp-1–VEGFR2 complex directly senses shear stress and regulates the site-specificity of atherosclerosis.Recently, Mehta et al.  revealedApoe−/− mice fed a high-fat diet, Sema7a deletion attenuates atherosclerotic plaque formation mainly in the aortic arch, an atheroprone region exposed to disturbed blood flow. Disturbed blood flow upregulates vascular endothelial Sema7A expression in the atheroprone lesser curvature. Sema7A enhances the endothelial expression of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 via β1-integrin, resulting in the promotion of leukocyte adhesion and plaque formation. Another study by Hu et al. [In 2018, Hu et al.  revealedu et al.  has reveu et al. . TherefoSemaphorin signaling has emerged as a pivotal factor regulating the pathogenesis of metabolic diseases by modulating immune responses, endothelial functions, and neural responses. Although recent advances have elucidated the importance of semaphorin signaling in metabolic disorders, several challenges remain in therapeutically targeting semaphorin signaling. First, semaphorin–plexin interactions are context-dependent and highly redundant, which makes it difficult to selectively target specific ligand-receptor interactions in semaphorin signaling. Second, most of the studies described herein utilized mice with whole body deletion of semaphorin molecules. Given that various types of cells express semaphorins and that their expression is tightly regulated during tissue development, this approach does not clarify the specificity of semaphorin signaling in the context of disease development. In summary, the translation of recent experimental insights into successful clinical interventions requires deciphering the spatiotemporal dynamics of semaphorin signaling, and developing reagents that selectively activate or inhibit semaphorin signaling.


Radiation-induced lung injury (RILI) is a form of radiation damage to normal lung tissue caused by radiotherapy (RT) for thoracic cancers, which is most commonly comprised of radiation pneumonitis (RP) and radiation pulmonary fibrosis (RPF). Moreover, with the widespread utilization of immunotherapies such as immune checkpoint inhibitors as first- and second-line treatments for various cancers, the incidence of immunotherapy-related lung injury (IRLI), a severe immune-related adverse event (irAE), has rapidly increased. To date, we know relatively little about the underlying mechanisms and signaling pathways of these complications. A better understanding of the signaling pathways may facilitate the prevention of lung injury and exploration of potential therapeutic targets. Therefore, this review provides an overview of the signaling pathways of RILI and IRLI and focuses on their crosstalk in diverse signaling pathways as well as on possible mechanisms of adverse events resulting from combined radiotherapy and immunotherapy. Furthermore, this review proposes potential therapeutic targets and avenues of further research based on signaling pathways. Many new studies on pyroptosis have renewed appreciation for the value and importance of pyroptosis in lung injury. Therefore, the authors posit that pyroptosis may be the common downstream pathway of RILI and IRLI; discussion is also conducted regarding further perspectives on pyroptosis as a crucial signaling pathway in lung injury treatment. Therapy for lung cancer risks producing adverse events, such as radiation-induced lung injury (RILI) and immunotherapy-related lung injury (IRLI). The incidence of RILI ranges from 5%-50% , but theTherefore, this review revisits previous works regarding the signaling pathways of RILI and IRLI and summarizes the potential crosstalk between RILI and IRLI. These signaling pathways have potential for clinical application as therapeutic targets. Moreover, in this review, the combination of radiotherapy and immunotherapy is considered and it is hypothesized that pyroptosis is likely a common downstream pathway of RILI and IRLI, a recognition that may facilitate and guide further research.RILI is a dose-limiting complication of radiotherapy for thoracic cancers and manifests as lung tissue damage, which is involved in acute radiation pneumonitis and chronic radiation pulmonary fibrosis. Many clinical trials and experiments in animal models have shown that a complex response after radiation leads to RILI –13; thisThese diverse signaling pathways exert different effects in epithelial cells, endothelial cells, fibroblasts, extracellular matrix molecules, and infiltrating immune cells. Below, we list three main signaling pathways of RILI in Many investigations have identified TGF-β as the key molecule in many human fibrotic conditions, and TGF-β promotes the process of fibrogenesis in tissue cells and the extracellular matrix –23. It iRoberts et al. found that Smad3 contributes to the pathogenic effects of TGF-β . This grHMGB1, an HMG protein, senses and coordinates the cellular stress response and functions as a damage-associated molecular pattern. HMGB1 exerts widespread biological effects throughout the body, including effects on metabolism, inflammation, immunity and cell death –36. In aThe nuclear factor erythroid 2 related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway is one of the most important mechanisms in the body’s defense against oxidative stress. Nrf2 is an original member of the mammalian cap ‘n’ collar transcription factor family with a highly conserved basic region-leucine zipper (bZIP) motif , and convia Nrf2  mice exposed to 20 Gy single-fraction thoracic radiation that migratory inhibition factor (MIF) may contribute to age-related susceptibility to thoracic radiation via Nrf2 . Similarvia Nrf2 . Howevervia Nrf2 . Duru etnt genes . Furthernt genes . All of In addition to the abovementioned signaling pathways, other possible pathways, such as melatonin-mediated miR-30e/nucleotide-binding domain-like receptor protein 3 (NLRP3), chemokine C-C motif ligand 2 (Ccl2), and Wnt/β-catenin signaling pathways, may play an important role in mediating RILI –65. ReceAdditionally, dosimetric factors play a critical role in RILI. There are few doubts that dosimetric parameters including irradiated volume, mean lung dose (MLD), dose delivered, schedule, and tumor location are risk factors for RILI , 70. AmoIn efforts to reduce dose exposure to by the normal lung, several new technologies have been recently developed. For instance, the magnetic resonance imaging linear accelerator (MR-Linac) provides more detailed real-time visualization of the tumor and surrounding tissue anatomy and facilitates the more precise use of adaptive re-planning . AdditioMany signaling pathways are activated under radiation stress, such as the main pathways introduced above. A common effect of these pathways is collagen aggregation. In the clinic, glucocorticoid drugs are the mainstay of RILI treatment. As the understanding of the mechanism of RILI has deepened, many drugs that can precisely target molecules in these signaling pathways or affect their common components have been identified. TGF-β is increasingly recognized for its role in the tumor environment. TGF-β not only can promote inflammation and fibrosis of the lung under radiation but also has an essential effect on tumor progression and escape . Many clImmunotherapy is a type of antineoplastic therapy based on mechanisms of the immune system aimed at facilitating immune function to kill cancer cells. Cancer immunotherapies can be divided into the following five classes: immune checkpoint inhibitors, lymphocyte-promoting cytokines, engineered T cells such as CAR T cells, agonistic antibodies against costimulatory receptors, and cancer vaccines. Among these classes, immune checkpoint inhibitors are the most commonly used . Many clThere are several reviews about the mechanisms of irAEs that inspire discussion about signaling pathways in IRLI –120. MorFirst, immune checkpoint inhibitors promote a shift from the exhausted T cell phenotype to an active effector phenotype, and generalized immune activation facilitates the production of cytokines due to T cell activation , 123. ReIn addition to these mechanisms that induce lung injury, other signaling pathways also contribute to IRLI. Recently, Jodai and coworkers reported the first case of acute eosinophilic pneumonia (AEP) as an irAE in a lung cancer patient who had received PD-1 blockade therapy . This stvia cytotoxic granules (perforin and granzymes), cytokines and Fas/FasL interactions while Th cells may secret IL-2, IL-6, IL-17, IFN-γ and TNF-α. Moreover, abnormal activation of B cells which may be activated by a T cell–independent mechanism can produce autoantibodies and induce antibody-dependent cell-mediated cytotoxicity. All of these three pathways can attack normal cells and cause cell death. Generally, the type of cell death caused by T and B cells is apoptosis which is regarded as silent cell death  that critically depends on the formation of plasma membrane pores by members of the gasdermin protein family, often  as a consequence of inflammatory caspase activation , 185. ItA considerable number of studies have addressed the key role of pyroptosis in lung injury , 194. AcPyroptosis is also closely related to IRLI. As mentioned above, normal lung tissue cells are attacked and damaged due to generalized immune activation from checkpoint neutralization, preexisting autoantibodies, and off-target effects of T cell-mediated immunity. With lysis of these  cells, double-stranded DNA and endogenous danger signals may be recognized by the AIM2 inflammasome and DAMPs may be recognized by the NLRP3 inflammasome. This may mediate pyroptosis of tissue cells leading to pneumonitis in IRLI, similar to the events of pyroptosis in RILI. IL-1β and IL-18, as the products of inflammasome activation, result in proinflammatory T cell differentiation and target organ damage , 202. MoUnfortunately, adverse events such as RILI and IRLI are not uncommon. More research and knowledge on the mechanisms of RILI and IRLI should be applied for the diagnosis and treatment of patients. Thus, more exploration and discovery to pinpoint the signaling pathways and mechanisms of RILI and IRLI are required. This review sheds new light on the crosstalk among these signaling pathways, which may provide targets for the prevention and treatment of RILI and IRLI. Furthermore, pyroptosis, a focus of frontier research, could be the key to elucidating mechanisms and solving problems including cancer and adverse events caused by cancer treatment.JY, DC, and MW contributed to the study design, manuscript discussion and critical revision. ZZ and JZ drafted the manuscript. XL assisted with the collection and visualization of the data. JY, DC and VV revised and polished the manuscript. All authors contributed to the article and approved the submitted version.The study was supported by funds from the Academic Promotion Program of Shandong First Medical University (2019ZL002) and the foundation of National Natural Science Foundation of China  and Science Foundation of Shandong .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A continuous increase in ambient temperature caused by global warming has been considered a worldwide threat. As sessile organisms, plants have evolved sophisticated heat shock response (HSR) to respond to elevated temperatures and other abiotic stresses, thereby minimizing damage and ensuring the protection of cellular homeostasis. In particular, for perennial trees, HSR is crucial for their long life cycle and development. HSR is a cell stress response that increases the number of chaperones including heat shock proteins (HSPs) to counter the negative effects on proteins caused by heat and other stresses. There are a large number of HSPs in plants, and their expression is directly regulated by a series of heat shock transcription factors (HSFs). Therefore, understanding the detailed molecular mechanisms of woody plants in response to extreme temperature is critical for exploring how woody species will be affected by climate changes. In this review article, we summarize the latest findings of the role of HSFs and HSPs in the HSR of woody species and discuss their regulatory networks and cross talk in HSR. In addition, strategies and programs for future research studies on the functions of HSFs and HSPs in the HSR of woody species are also proposed. As one of the most harmful abiotic stresses, heat stress poses a threat to plant life by impacting plant metabolism directly  and other greenhouse gases. In addition, woody plants are the main biomass resources for biofuels. Due to the perennial and long life cycle, woody plants experience more severe and extreme abiotic stresses during their lives compared to herbaceous plants. Woody plants may have evolved more complex stress-responsive mechanisms , HSP40s, HSP60s, HSP70s, HSP90s, and HSP100s  chaperons with a molecular mass of 15–30 kDa. According to the sequence homology and subcellular localization, sHSPs are classified into 11 different classes. Class I–VI sHSPs are localized in the nucleus or cytoplasm, and the rest five classes of sHSPs are localized in mitochondria, chloroplasts, peroxisomes, or ER      , yeast, and Arabidopsis , and inducing the expression of heat-responsive (HR) genes , responds to high temperature, salt, and drought stresses and has a certain effect on the adaptability of introduction and domestication  cytoplasmic class III sHSP gene (PmHSP17.9) improves the thermotolerance of transgenic Arabidopsis by enhancing superoxide dismutase (SOD) activity  sHSP gene JrsHSP17.3 in leaves enhances tolerance to cold, heat, and salt stresses by scavenging the accumulation of ROS and by accumulating osmotic adjustment substances  from C. sinensis could be highly induced by heat and cold stresses. Overexpression of these small CsHSPs confers heat and cold tolerances in transgenic Pichia pastoris (P. pastoris) and Arabidopsis -dependent and ABA-independent signaling pathways and other stress signaling pathways -localized sHSP, sHSP22, is involved in ABA and auxin signaling crosstalk ] increases the heat sensitivity, which is manifested in the higher accumulation of ROS and lower membrane stability in silenced plants , hydrogen peroxide (H2O2), and putrescine    ] ]  ]  BpHSFA4 gene in leaves improves the salt stress tolerance by increasing the ability to scavenge ROS, thereby reducing cell damage or cell death and enhancing birch resistance    motif is commonly found in the promoters of HSF target genes  and HSFA2 (PtHSF-A2), which is consistent with their protein–protein interactions in Arabidopsis and tomato (Meiri et al., Plants possess a complex HSR regulatory network consisting of multiple HSF and HSP genes during HSR in woody plants is still lacking. On the one hand, the transcriptional regulation or interaction between HSF and HSP in woody plants is unclear. Zhang et al. (P. trichocarpa natural variant populations to successfully identify the upstream regulatory transcription factor WRKY that controls the expression of the HCT2 gene. This strategy provides a new method to identify the transcriptional regulatory relationship between genes. Therefore, the public gene expression database of woody plants can be used to preliminarily examine whether there is a potential regulatory or interaction relationship between HSFs and HSPs in these species. In addition, the continuous maturity of ChIP-Seq, CUT&Tag-Seq, and other technologies have effectively improved the ability to analyze the interaction between protein and DNA. The recently developed reverse-ChIP technology (Wen et al., HSF or HSP genes obtained from woody species in the model plant Arabidopsis or yeast. Therefore, fast and efficient gene function validation systems are urgently needed. The protoplast transient expression system (Zhang et al., With ongoing greenhouse gas emissions, the ambient temperature is expected to rise over time. Woody plants have evolved more complex stress response mechanisms to cope with changing environments than herbaceous plants to guarantee a longer lifespan (Anderegg et al., g et al.  used theJZ conceived the study. JZ, FT, and X-LH drafted the manuscript. TY, XY, J-GC, and M-ZL revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Among human genetic diseases, Fanconi Anemia (FA) tops all with its largest number of health complications in nearly all human organ systems, suggesting the significant roles played by FA genes in the maintenance of human health. With the accumulated research on FA, the encoded protein products by FA genes have been building up to the biggest cell defense signaling network, composed of not only 22+ FA proteins but also ATM, ATR, and many other non-FA proteins. The FA D2 group protein (FANCD2) and its paralog form the focal point of FA signaling to converge the effects of its upstream players in response to a variety of cellular insults and simultaneously with downstream players to protect humans from contracting diseases, including aging and cancer. In this review, we update and discuss how the FA signaling crucially eases cellular stresses through understanding its focal point. Fanconi anemia (FA) is a rare human genetic disorder with an incidence rate of about one in 136,000 births, and half of FA patients are diagnosed prior to age 10 ,3,4,5. FIt is unfortunate to say that health complications of FA have evidently indicated the functional significance of FA or FA-related genes. To date, the fact that FA patients who live into adulthood are likely to develop AML or a variety of solid tumors, has led to an exciting field of cancer research. Under the circumstance, understanding these FA or non-FA gene functions has become a new direction to advance cancer research. Indeed, many studies including ours have demonstrated numerous discoveries in advancing our understanding of cancer prevention, etiology, and treatment. Noting many published reviews on the relation between FA signaling and human diseases ,5,6,9,10FA signaling is defined upon similar clinical, cellular, and molecular abnormalities displayed from at least 22 groups of FA patients. FANCD2 and its paralog FANCI stand at the center and become the focal point of this huge signaling network . The monThe FA complementation groups currently include FANCA , FANCB, FANCC, FANCD1 , FANCD2, FANCE, FANCF, FANCG , FANCI, FANCJ , FANCL, FANCM and FANCN , FANCO , FANCP , FANCQ , FANCR , FANCS , FANCT , FANCU (XRCC2), FANCV , and FANCW  [FANCD2 (and FANCI) monoubiquitination leads to the retention of the ID complex at sites of stalled replication or the damaged DNA. FA cells are exquisitely sensitive to agents that increase stalled replication or DNA damage, such as UV, platinum, or mitomycin C (MMC) ,13,14,15FANCD2 is monoubiquitinated in response to DNA damage, resulting in its localization to nuclear foci with FANCS, FANCD1, and others involved in homology-directed DNA repair, nonhomologous end joining, postreplication repair, etc. Accumulated studies indicate that FANCD2 acts in coordination with many repair proteins known and/or yet to be identified in nearly all phases of the DNA damage responses, sensing, signal transduction, and execution of repair . As suchFA cells have high incidences of aneuploidy and micronucleation, often occurring as a result of chromosome missegregation . In addiWhile fathoming the functional varieties of FANCD2, we started to characterize an unrecognized form of FANCD2, namely, FANCD2-V2 in contrast to the long-known one, FANCD2-V1 . This wap < 0.01) in tumor samples than the matched normal tissues by analyzing publicly available datasets . This discovery represents another important biologic variation that may be capable of marking nonmalignancy from malignancy. Our following studies also showed that the expression patterns of FANCD2-V1 and V2 are involved in the use of an alternative polyadenylation site (APS), which is regulated by DNA methylation in the distal or proximal regions of APS (Me-D and Me-P) . The ratTo better understand how Me-D/Me-P and V1/V2 derived from FA singling to be promising biomarkers, their biological bases need to be further determined. We reported that TRK-Fused Gene (TFG) was a specific functional partner for FANCD2-V2 in early cellular responses to DNA damage, but not for FANCD2-V1. As such, FANCD2-V2 forms nuclear foci upon DNA damage, and both its focus appearance and disappearance are earlier than FANCD2-V1 . HoweverAmong the listed 22 FA complementation groups, FANCI was discovered as a relatively new complementation group of FA, which acts as a paralog of FANCD2 required for DNA repair ,43. It sThe above functional highlights for FANCI demonstrate its extreme importance across a variety of cellular processes. This was further strengthened by numerous FANCI variations directly associated with many human cancers. Its germline mutations were found in AML  or relatThe FA signaling converges on its focal point (the ID complex), which is not only a substrate for the FA core complex but also a potential platform for recruiting downstream FA signaling players, including nucleases and other FA and non-FA proteins for DNA damage repair ,54 Figu. SignifiCurrently, the structure study provides much in-depth insight into protein–protein interactions, as such an interaction of FANCD2 and FANCI is attributed at least to armadillo (ARM) repeats and an EDGE motif at the C-terminus of FANCI . A centrGermline FA gene mutations have been directly associated with many cancers including breast, ovarian, and pancreatic cancer owing to the defects relating to FANCD1/N/C and/or/G ,69,70,71Somatic inactivation of the FA pathway could stem from the impairment of any FA proteins. Among these, the hypermethylated FANCF promoter, resulting in a compromised FA signaling, was found in 6.7% to 30% of tested tumor cell lines including testis, lung, ovarian, and cervical cancer lines ,74,75. RIn our earlier studies, we discovered that a new form of FANCL protein, named FAVL, dysregulates the FA signaling in non-FA human tumor cells and acts as a tumor promoting factor by inactivating FANCD2 and FANCI/the ID complex ,80,81,82To date, the functional significance of germline variations in FA genes has been frequently reported ,87. For For many FA or FA-related proteins upstream of the ID complex, their variants will, to some extent, impair the monoubiquitination of FANCD2 and FANCI and thus the tumor suppressive roles of FA signaling. For example, FANCL has many variants, such as the known substrate binding mutants , an FA mutation (R221C), and 14 cancer-associated mutations  ,91,92 anHawaii Cancer at a Glance, 2012–2016), we decided to explore possible biological variations that are unique to Native Hawaiians with colorectal cancer (CRC). The RNA-seq on the 40 CRC tumor and matched normal samples  were used to establish the first resource of The Cancer Genome Atlas (TCGA)-like datasets specifically for Native Hawaiians (NH) or Pacific Islanders (PI). Using this first-hand resource, we identified biological variations unique in NH/PI that impair FA signaling, which is not part of canonic causes known during colorectal oncogenesis. Now, we will follow this novel lead in order to uncover how FA signaling contributes to race-specific causes of CRC. As a consequence, our studies may promise valuable insights into CRC susceptibility and targets of intervention with the goal of positively impacting CRC health disparities, particularly those associated with NH/PI. Our ongoing work indicates FA signaling is specifically involved in a novel aspect of cancer research and cancer health disparities . In recoA large body of research demonstrates that FA signaling is of extreme importance to facilitate an understanding of the pathogenesis of human diseases. Noting the indefinite nature of upstream and downstream FA signaling, its relatively defined center appears more capable of helping understand how FA signaling functions as a unique biological/genetic model system, which is a naturally existing advantage for biomedical researchers to take. We updated the functional varieties of FANCD2 and FANCI, the two players at the center of FA signaling. Both have well-known functioning upon DNA damage or during DNA replication, but also play important roles in the other phases of a cell cycle under both stressed and nonstressed conditions by participating in many biological processes, including RNA spacing, apoptosis, etc., aside from DDR. When FANCI or FANCD2 is not properly functioning, both can lead eventually not only to cancer as emphasized herein but also to aging and many other states, including metabolic disorders . Despite our emphasis on the functional roles of FANCD2 and FANCI in maintaining genome stability, it remains unclear how exactly FANCD2 and FANCI promote chromosomal stability when replication forks encounter abnormal DNA structures or tightly chromatin-bound proteins when probing the in-depth molecular insights into replication. Upon activation of the master replication checkpoint kinase ATR, FANCD2 and FANCI accumulate within chromatin in the vicinity of replication forks, and FANCD2 associates transiently with the replication machineries. It is also unclear as to the in-depth molecular insights into DDR in the maintenance of genome stability, which are attributed to FANCD2 and FANCI. FANCD2 may bind directly to histones and promotes the nucleosome assembly upon DNA damage or during DNA replication. Combined, these suggest that FANCD2 and FANCI regulate the further molecular machineries for chromatin-based processes and many others, preventing the human genome from going awry for cancer and other human diseases. With constant growth in its upstream and downstream, FA signaling emerges to be the biggest cellular defense-signaling network. However, we are able to appreciate its functional roles through the relatively defined axis, its focal point (FANCD2 and FANCI). FA signaling is involved in both DDR-dependent and independent cellular processes under stressed and nonstressed conditions. Consequently, FA signaling protects humans from a variety of diseases beyond cancer. Among FA genes, FANCD2 is the most conserved from bacteria to humans ,97. ThisIn a study of molecular tumorigenesis of thymoma , the autTo date, numerous targets derived from either upstream or downstream of DDR/FA signaling are under testing to increase the sensitivity of chemotherapy ,103,104.


Uric acid (UA) is the end metabolic product of purine metabolism. Early on, UA was considered to be a metabolite with a certain antioxidant capacity. As research has progressed, other properties of UA have been explored, and its association with many diseases has been found. The association between UA and kidney disease and cardiovascular disease is well established; however, there is still a paucity of reviews on the association between UA and the female reproductive system. An increasing number of epidemiological studies have shown elevated serum UA levels in patients with polycystic ovary syndrome (PCOS), endometriosis, etc. Additionally, serum UA can be used as a predictor of pregnancy complications and adverse foetal outcomes. An increasing number of animal experiments and clinical studies have revealed possible mechanisms related to the involvement of UA in certain female reproductive disorders: oxidative stress, chronic inflammation, mitochondrial dysfunction, etc. This article reviews the current mainstream mechanisms regarding the pathogenesis of UA and the role of UA in certain specific female reproductive disorders (direct involvement in the development of certain diseases or enhancement of other risk factors) in the hope of contributing to clinical prevention, diagnosis, treatment and improvement in prognosis. Previous studies have revealed that physiological levels of uric acid (UA) have a protective function in vivo due to their antioxidant effect . However. XOR is the key enzyme in this process and has two convertible forms: xanthine dehydrogenase (XDH) and XO. XDH can convert NAD + to NADH via FADH2, while XO can convert O2 to hydrogen peroxide and free radical superoxide anion (O2.−) via FAD [UA is a major product of purine metabolism catalysed by xanthine oxidoreductase (XOR). Plasma UA levels can be increased by exogenous pathways such as excessive intake of high-purine and high-fructose foods and are mainly affected by catabolism from endogenous pathways such as those in the liver and small intestine . HyperurFig. . XOR is  via FAD . Thus, X via FAD . The mai via FAD .Fig. 1TUA is also found in the follicular fluid of the female ovary. Studies on buffalo ovaries have proven that disruption of the plasma-follicular barrier structure is associated with increased levels of UA . In addiUA is a known antioxidant and is a major contributor to antioxidant potential in vivo. UA not only removes singlet oxygen and free radicals but also reduces the damage of peroxynitrite to proteins involved in nearly half of the antioxidant effects in vivo . UA has Oxidative stress is related to the pathogenesis of many diseases, including cardiovascular disease, cancer, and neurodegenerative diseases , 17. TheIn addition to inducing oxidative stress, high concentrations of serum UA also affect lipid synthesis and lipid oxidation distortion. High levels of UA stimulate the NADPH oxidase isoform NADPH oxidase 4 (NOX4) in mitochondria, which increases superoxide production and consequently decreases aconitase activity. This leads to the accumulation of its substrate citric acid, promoting its re-catabolism to acetyl-CoA for lipid synthesis . ExcessiAbnormal lipid metabolism, on the one hand, produces lipotoxicity, which has a direct adverse effect on cells, and on the other hand can produce large amounts of ROS, which induces stress damage to intracellular organelles leading to cell death , 22. NakHigh levels of UA can lead to systemic sterile inflammation and may also become a pro-inflammatory factor. The mainstream view is that high levels of UA form monosodium urate (MSU) around cells through extracellular sodium reactions. Apoptosis-associated speck-like protein containing a caspase-associated recruitment domain (ASC) recruits caspase-1 and, together with NLR family pyrin domain containing 3 (NLRP3), forms NLRP3 inflammatory vesicles that convert MSU-produced pro-IL-1 into IL-1β, which triggers the inflammatory response . HoweverIn addition to promoting inflammation in the form of MSU crystals, soluble UA in adipocytes can also mediate lipid oxidation by activating mitogenic protein kinases such as p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated kinase 1/2 (ERK1/2) through NADPH oxidase activation . In othe.UA serves as an end product of purine metabolism reflecting the metabolic state of the body, and it can maintain the level of oxidation in the body under physiological conditions. However, excessive accumulation of UA can cause damage to multiple systems throughout the body. In reproductive system diseases, although these mechanisms are still unclear, we currently believe that UA may be involved in key aspects of oxidative stress, inflammation, and metabolic disorders in vivo, leading to the development of various pathological conditions Fig. .Fig. 2HReproductive hormones also affect serum UA levels. Although there is considerable disagreement on the issue of specific hormones and UA levels, the prevailing view is that high levels of testosterone are strongly associated with elevated serum UA levels and hyperuricaemia. Testosterone may induce functional alterations in the renal UA reabsorption system through increased expression of URAT1, sodium-coupled monocarboxylate transporter 1, and/or 2  (SMCT1 and/or 2) or GLUT9, among others . Animal 2), and ethanol intake [The link between oestradiol and UA is controversial. High UA levels are observed in menopausal women under normal conditions and may also be associated with postmenopausal insulin resistance (IR), obesity . IL-1β is produced when UA forms inflammasomes (mentioned above). Serum IL-1β levels were significantly higher in women with deep infiltrating endometriosis than in normal women in the control group [Endometriosis is considered to be an inflammatory, immune, and haemorrhagic disease with the main clinical manifestations of dysmenorrhea, chronic pelvic pain, menstrual abnormalities, and infertility. Accumulating evidence suggests that IL-1β plays an important role in the development of endometriosis Fig. . IL-1β iol group . IL-1β mol group , 65. Akool group  reportedol group . In addiol group . This maWomen with endometriosis often have prolonged, severe pelvic pain that is not relieved by analgesic drugs. This may be because IL-1β increases brain-derived neurotrophic factor (BDNF) and the nerve injury-inducible protein 1 (Ninj1) through a signal transduction pathway mediated by c-Jun N-terminal kinase (JNK) and NF-κB, which in turn stimulates the associated nerves and causes pelvic pain , 70. It Up to approximately one-half of women with endometriosis have infertility . It has In the early stages of normal pregnancy, serum UA levels decrease due to increased plasma volume, increased renal clearance, and the urate excretion promoting effect of oestrogen. Serum UA stabilizes at a certain level during most of the second trimester. In late gestation, serum UA increases rapidly due to the excretion of foetal metabolites through the mother . Almost HDPs include four conditions: gestational hypertension, PE, chronic hypertension and chronic hypertension with superimposed PE. PE is a severe HDP that begins with impaired maternal uteroplacental vascular formation. If not treated early, it may progress to eclampsia as well as maternal and foetal in utero growth restriction and death. Many pregnant women with PE commonly have elevated UA levels or even hyperuricaemia. There are multiple potential sources of increased UA in PE, the main ones are as follows , 78: (i)Similarly, in PE patients, high UA levels affect the normal function of blood vessels and the placenta mainly through oxidative stress and inflammation. UA induces oxidative stress, which is considered to be a key link in PE. On the one hand, oxidative products promote the production of vasoconstrictive substances, activating the local renin-angiotensin system in human endothelial vascular cells including angiotensin II. On the other hand, it leads to decreased production and bioavailability of diastolic substances  inducing endothelial dysfunction . DoughanHigh UA levels also have a profound effect on endothelial cell migration, proliferation, and apoptosis. UA significantly inhibited serum-induced proliferation in human umbilical vein endothelial cells and their migration . At the GDM is a type of diabetes that is not present before pregnancy and occurs during pregnancy. Although it may disappear after delivery, women who have had GDM are at an increased risk of developing type 2 diabetes later. Early pregnancy UA levels can be used to some extent as a secondary predictor of GDM and may be partially involved in the development of GDM . High UA(i) The direct toxic effects of UA on foetal growth. (ii) Elevated UA levels can affect the transport of nutrients. For example, UA-induced oxidative stress can inhibit mammalian target of rapamycin (mTOR) signalling and reduce amino acid transport to the foetus [(iii) Finally, elevated UA levels can also affect hormone levels in foetuses, especially growth hormones [Serum UA in some cases can directly affect foetal growth. In addition to oxidative stress and inflammation affecting the vasculature and placenta, as mentioned earlier, other causes include the following: e foetus . (iii) Fhormones .Serum UA levels have also been associated with the risk of preterm birth in pregnant women. In a case-control study by Roberts et al. , hyperurThe relationship between UA and inflammation has already been mentioned above, including the involvement of multiple inflammatory factors and the formation of inflammatory vesicles. Several studies have found that elevated IL-1β levels may increase the risk of preterm birth. The evidence that IL-1β increases the risk of preterm birth is as follows: (i) IL-1β expression is increased in the amnion, chorionic villus, cervix, and myometrium after normal delivery . IL-1β cElevated serum UA levels are considered to be a predictor of maternal and infant complications, so early measurement of serum UA may be beneficial in the diagnostic treatment of this clinical syndrome. The sensitivity from the study by Bellos et al.  for predAmniotic fluid UA monitoring is also considered to be of value in predicting foetal growth. In early gestation, amniotic fluid is mainly derived from the plasma component of the embryo. As the organs of the embryo begin to mature and develop, the metabolites of the foetus gradually increase, as do the levels of amniotic fluid UA. It has been suggested that amniotic fluid UA is an important predictor of infant birth weight in mid-gestation . In addiIn this review, we tried to summarize the relationship between UA and female reproductive diseases, focusing on some of the characteristics of UA and its possible mechanisms in the development of PCOS, endometriosis, pregnancy complications, and other diseases. We believe that there is a close interaction between abnormal UA levels in patients with these diseases and various symptoms and long-term complications. We suspect that there is a certain correlation between UA levels and oxidative stress, inflammation, mitochondrial dysfunction, and so on. These factors influence and promote each other, forming a vicious circle. We hope our results can be helpful in showing possibilities for potential prevention, treatment, and prognostic assessment.Our understanding of the role of UA in the reproductive system is also growing as more literature is added. Serum UA levels have been proposed to predict the occurrence of PE and adverse maternal and foetal outcomes . A model