Andrographis paniculata and exhibits activity against several viruses, including Epstein–Barr virus (EBV). However, the particular mechanism by which andrographolide exerts an anti-EBV effect in EBV-associated gastric cancer (EBVaGC) cells remains unclear. We investigated the molecular mechanism by which andrographolide inhibits lytic reactivation of EBV in EBVaGC cells (AGS-EBV cell line) using proteomics and bioinformatics approaches. An andrographolide treatment altered EBV protein-expression patterns in AGS-EBV cells by suppressing the expression of EBV lytic protein. Interestingly cellular transcription factors (TFs), activators for EBV lytic reactivation, such as MEF2D and SP1, were significantly abolished in AGS-EBV cells treated with andrographolide and sodium butyrate (NaB) compared with NaB-treated cells. In contrast, the suppressors of EBV lytic reactivation, such as EZH2 and HDAC6, were significantly up-regulated in cells treated with both andrographolide and NaB compared with NaB treatment alone. In addition, bioinformatics predicted that HDAC6 could interact directly with MEF2D and SP1. Furthermore, andrographolide significantly induced cell cytotoxicity and apoptosis of AGS-EBV cells by induction of apoptosis-related protein expression. Our results suggest that andrographolide inhibits EBV lytic reactivation by inhibition of host TFs, partially through the interaction of HDAC6 with TFs, and induces apoptosis of EBVaGC cells.Andrographolide is the principal bioactive chemical constituent of  EBV is a ubiquitous gamma-herpesvirus and is strongly associated with the development of several human cancers, including Burkitt’s lymphoma (BL), Hodgkin’s lymphoma, nasopharyngeal carcinoma (NPC), and EBVaGC. EBVaGC is a subtype of gastric adenocarcinoma (GC) and accounts for approximately 10% of all GCs. EBV establishes two alternative modes of infection in the host cell, latent and lytic infection. Latent infection plays an essential role in carcinogenesis through the activities of latent-stage proteins, especially LMP1. Upon reactivation induced by various stimuli, EBV undergoes three consecutive lytic stages, the immediate early (IE), early (E), and late (L) stages. The IE proteins, Zta and Rta, activate transcription from the promoters of E genes, which trigger EBV genomic DNA replication from the origin of replication. Then the L genes that encode structural proteins are expressed, followed by viral genome packaging into infectious virion particles. In recent years, the EBV lytic cycle has been reported to contribute to carcinogenesis of several cancer types [BZLF1 promoter (Zp) and BRLF1 promoter (Rp) [The regulation of EBV lytic reactivation is initially determined by the availability of viral proteins and host TFs that can either suppress or activate the ter (Rp) ,3. Zp isAndrographis paniculata (Burm. f.) Nees (family Acanthaceae) contains diverse compounds such as labdane diterpenoid lactones, flavonoids, and miscellaneous compounds and is widely used as a traditional medicine in Asian countries [Andrographolide exhibits various medicinal properties, such as anti-inflammatory, antioxidant, anti-bacterial, anti-retroviral, anti-tumor, anti-diabetic, anti-malarial, anti-hypertension, anti-rheumatism, hepatoprotective, immunostimulant, anti-cancer, anti-angiogenic, and anti-viral activities ,5,6. Andountries . Anti-viountries , dengue ountries , chikungountries , human pountries , hepatitountries , and infountries .Andrographolide can inhibit EBV lytic reactivation by suppressing EBV lytic genes and the activity of Zp . HoweverThe GC cell lines, AGS and AGS-EBV  were cultured in RPMI 1640 medium containing 10% FBS and antibiotics (streptomycin and penicillin). The andrographolide compound was prepared as previously described  by the D5 cells/mL) were seeded into 96-well plates and pre-incubated at 37 °C, 5% CO2 for 24 h. After incubation, culture medium was removed, and cells were subjected to one of three treatments: andrographolide alone, NaB alone, or a combination of andrographolide and NaB. Cells were then incubated at 37 °C, 5% CO2 for 24 and 48 h. Cell viability was determined using the CCK-8 assay [To investigate whether andrographolide induces cytotoxicity in GC cell lines. AGS and AGS-EBV cells (1 × 10-8 assay .25) or concurrently treated with NaB and andrographolide at CC25. A control group did not receive either treatment. Total protein was extracted using TRIzol™ reagent  according to the manufacturer’s instructions. The total protein concentration was determined using Bio-Rad protein assay-dye reagent concentration kit. Protein samples were analyzed on 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE).The EBV-infected cell line was treated with andrographolide at 25% cytotoxic contraction (CCProtein content was determined using the Lowry method  and bovim/z) 150–2200 .The tryptic peptide samples were prepared for injection into an Ultimate 3000 Nano/Capillary LC System  coupled to a Hybrid quadrupole Q-Tof impact II™  equipped with a nano-captive spray ion source. Briefly, peptides were enriched on a µ-precolumn 300 µm i.d. × 5 mm C18 Pep Map 100, 5 µm, 100 A , separated on a 75 μm I.D. × 15 cm, and packed with Acclaim Pep Map RSLC C18, 2 μm, 100 A, nanoViper . Solvent A and B containing 0.1% formic acid in water and 0.1% formic acid in 80% acetonitrile, respectively, were applied on the analytical column. A gradient of 5–55% of solvent B was used to elute the peptides at a constant flow rate of 0.30 μL/min for 30 min. Electrospray ionization was carried out at 1.6 kV using the Captive Spray. Mass spectra (MS) and MS/MS spectra were obtained in the positive-ion mode over the range  [https://string-db.org/, accessed on 24 August 2021) [The functional annotation of the unique proteins was performed by PANTHER . The difhttps://zlab.umassmed.edu/bu/rama/, accessed on 29 November 2021). All ionizable amino acid residues in predicted models were protonated at pH 7.4 by using H++ 1.0 . Molecular docking simulation was performed by the web-based service ClusPro [To further confirm protein–protein interaction, molecular docking was performed. The sequences of candidate proteins were submitted to SWISS-MODEL , protein ClusPro  to ident ClusPro .MEF2D and SP1) and HDACs (HDAC6 and HDAC9) was determined by qRT-PCR using SsoAdvancedTM SYBR® Green Supermix  in the QuantStudio 6 Flex Real-Time PCR System . The GAPDH gene was detected as an internal control. The primer sequences used in the present study are listed in Total RNA was extracted from cells that were treated for 24 h with either NaB or andrographolide or a combination of both using TRIzol™ reagent  according to the manufacturer’s instructions. The expression of host TF genes (5 cells/well) were seeded into a 6-well plate and incubated at 37 °C, 5% CO2 for 18 h to allow the cells to adhere to the plate. Cells were then either treated with NaB or andrographolide or a combination of both. Cells were then incubated at 37 °C, 5% CO2 for 18 h. After incubation, cells were collected by trypsinization and centrifugation. Apoptotic cells were detected using Dead Cell Apoptosis Kits  using flow cytometry , and data were processed using FlowJo software . Cells treated with 50 nM of staurosporine and 0.1% DMSO were used as positive controls and negative controls, respectively.The effect of andrographolide on cell apoptosis was analyzed using flow cytometry. Cells of the AGS-EBV and AGS cell lines . Real-time PCR data was evaluated using independent sample t-tests. Differences were considered statistically significant at a 25, 34.40 μM for 3 h, and subsequently incubated with or without NaB for 48 h. Total proteins were extracted and subjected to LC–MS/MS. As shown in To confirm whether andrographolide suppresses EBV lytic reactivation, AGS-EBV cells were pre-treated with andrographolide at CCThe functional annotation of proteins that were only expressed in cells subjected to concurrent treatment of andrographolide and NaB was done using PANTHER. These differentially expressed proteins were involved in biological processes, such as cell-surface receptor signaling pathway, cell communication, signaling transduction, and others B. In terPreviously, we found that andrographolide could inhibit EBV lytic reactivation by inhibition of EBV lytic protein expression and of viral particle production. In the present study, we investigated this further and found that expression of EBV lytic proteins, including BALF4, BALF5, BMRF1, BBLF3, BRLF1 and BZLF1 was significantly inhibited by andrographolide A In addiEmerging evidence demonstrates that various host TFs could regulate lytic reactivation of EBV. Therefore, we examined the expression of host TFs using LC–MS/MS. We found that the expression of CREB3L2, MEF2D, SP1, XBP1 and XPB was decreased in AGS-EBV cells treated with the combination of andrographolide and NaB compared with NaB treatment alone. Whereas the expression of CEBPA was increased in AGS-EBV cells treated with the combination of andrographolide and NaB compared with NaB treatment alone B. In addMEF2D and SP1) and suppressors (HDAC6 and HDAC9) of EBV lytic replication was determined by qRT-PCR. Similar to protein levels, the expression of MEF2D and SP1 was significantly decreased in AGS-EBV cells treated with andrographolide and NaB compared with NB-treated AGS-EBV cells  using the PPI prediction tool, PSOPIA, and found that the probabilities that HDAC6 directly interacts with MEF2D and SP1 were 99.22% and 34.17%, respectively. In case of HDAC9, the probabilities were 99.91% and 90.11%, respectively  of andrographolide in an AGS-EBV cell line [Previously, we determined cytotoxic concentrations  , influenBZLF1 [As mentioned earlier, EBV has two infection phases: lytic and latent. EBV lytic reactivation is mediated by host TFs that can repress or stimulate transcription of Zp and Rp. The Zp promoter contains the cis-acting motifs required for Zp activation. Host TFs, including MEF2D, SP1, SP3, CREB, ATF-1, the ATF-2/c-jun heterodimer, and AP1, can bind specifically to their responsive elements and activate Zp ,32,33,34BZLF1 .Consistently with this, we observed down-regulation of several host TFs, including CREB3L2, MEF2D, SP1, XBP1, and XPB, in AGS-EBV cells treated with andrographolide and in which lytic reactivation was subsequently induced by NaB compared with NaB treatment alone. In contrast, the expression of epigenetic mechanisms, including DNA methylation (via DNMT3A), histone acetylation (via HDAC6 and HDAC9), and histone methylation , were significantly increased in AGS-EBV cells treated with the combination of andrographolide and NaB when compared with NaB treatment alone. Therefore, the inhibition of EBV lytic reactivation may be mediated by epigenetic mechanisms.Accumulating evidence suggests that histone modification plays an important role in the inhibition of EBV lytic reactivation. The alteration of histone acetylation at the promoter for the BZLF1 gene determines the potential for EBV lytic reactivation . TreatmeIn addition to histone modification, HDACs may directly bind with host TFs. In this study, by using a protein–protein interaction prediction approach (PSOPIA), we found that HDAC6 and HDAC9 can directly interact with MEF2D and SP1. This agrees with studies showing that other HDACs can directly interact with MEF2D or SP1. SP1 and SP3 directly bind to specific consensus GC-rich sequences in the HDAC4 promoter to drive HDAC4 transcription . Sp1 is In the present study, we assessed the ability of andrographolide to induce cytotoxicity in the gastric cancer cell lines AGS and AGS-EBV. As expected, andrographolide combined with NaB induced cytotoxicity in both AGS and AGS-EBV cells when compared with NaB treatment alone. In addition, viability of AGS-EBV cells that were treated with andrographolide was significantly lower than for AGS cells. Consistent with this, andrographolide significantly induced apoptosis in gastric cancer cell lines, especially AGS-EBV. Previous studies have demonstrated that andrographolide can induce cell-cycle arrest and apoptosis of cancer cells by inhibiting cellular pathways such as phosphoinositide 3-kinase/protein kinase B, mitogen-activated protein kinase, and other tumor growth pathways, depending on the type of treated cells ,48,49. IIn this present study, we investigated the effects of andrographolide on the inhibition of lytic reactivation of EBV and induced apoptosis in gastric cancer cells (in vitro), which cannot be clearly clinically linked. In previous reports, EBV lytic reactivation could induce cellular genomic instability by induction of micronuclei and γH2AX expression, markers of DNA damage and genomic instability, and enhancement of the tumorigenicity of NPC ,56 and wThis study demonstrates for the first time that andrographolide can inhibit EBV lytic reactivation by modulating the expression of viral and cellular proteins, especially TFs (SP1 and MEF2). The expression of TFs can probably be regulated via processes including DNA methylation, histone acetylation, and histone methylation. Interestingly, HDAC6 can directly interact with TFs, which inhibit the transcriptional activity of these TFs. In addition, andrographolide also induces cytotoxicity and apoptosis in GC and EBVaGC cells.


Arabidopsis thaliana, we identified AT5G06830/C53, a soluble protein that directly interacts with ATG8. Biochemical and biophysical characterization of C53-ATG8 interaction using both human (CDK5RAP3) and Arabidopsis proteins revealed that C53 binds ATG8 via shuffled Atg8-family interacting motifs (sAIMs) located at its intrinsically disordered region (IDR). C53 is recruited to phagophores, precursors to autophagosomes, during ER stress in an autophagy-dependent manner. Consistently, c53 mutants are highly sensitive to ER stress treatments. C53 senses ER stress by forming a tripartite receptor complex that involves UFL1, the E3 ligase that mediates ufmylation, and its ER-resident adaptor protein DDRGK1. C53 activity is regulated by another ubiquitin-like protein, UFM1, which is transferred from C53 to the ribosomes upon ribosome collision/stalling at the ER, thereby activating the C53 pathway to recycle stalled nascent chains. Altogether our findings suggest C53 forms an ancient quality control pathway that links ribosome-associated quality control with selective autophagy at the ER.Reticulophagy, the autophagic degradation of the endoplasmic reticulum, is crucial to maintain ER homeostasis during stress. Although several reticulophagy receptors have been discovered recently, most of them have been studied using nutrient starvation. How macroautophagy/autophagy cross-talks with other ER-quality control mechanisms is largely unknown. Using ATG8-based affinity proteomics in the model plant  Accumulation of unfolded proteins in the ER triggers a series of quality control mechanisms such as the unfolded protein response/UPR and ER-associated degradation/ERAD. Recent studies have shown that reticulophagy, the recycling of ER compartments by selective autophagy, is also a major quality control mechanism that remodels the ER during starvation and stress.The endoplasmic reticulum is a highly dynamic network that is crucial for protein folding and maturation, lipid metabolism, and CaSince the discovery of the first reticulophagy receptors in yeast and mammalian cells in 2015, several other receptors have been identified. In mammals, these include ER-resident proteins RETREG1/FAM134B, RTN3L, SEC62, CCPG1, TEX264, ATL3, and the soluble protein CALCOCO1. Similarly, in plants, ATI1, ATI2, ATI3, RTN1, and ATSEC62 have been linked to ER recycling. Whether these receptors work in concert and how reticulophagy is intertwined with other ER-quality control pathways still remain largely obscure. In addition, mechanisms of activation of these reticulophagy receptors, and how they are targeted to the regions of the ER that needs to be recycled need further investigation.Unlike most other reticulophagy receptors, C53 is highly conserved in multicellular eukaryotes. This protein binds ATG8 via distinct binding epitopes, located at the C53 IDR, that we named shuffled AIMs (sAIMs). Stoichiometry analysis revealed that C53 binds two ATG8 proteins with a low micromolar binding affinity. Binding more than one ATG8 could help in clustering the core autophagy machinery to facilitate the reticulophagy process. In addition, C53 also interacts with RB1CC1/FIP200 and Atg11 respectively, similar to other reticulophagy receptors such as CCPG1.C53/CDK5RAP3 forms a receptor complex with UFL1, the E3 ligase that mediates ufmylation, and its adaptor protein, DDRGK1. UFL1/At3g46220 and DDRGK1/At4g27120 are co-delivered to the vacuole with C53 and are essential for C53-mediated autophagy. Consistently, c53, ufl1, ddrgk1, and the ufmylation pathway mutants are highly susceptible to ER stress treatments. Recently, other reticulophagy receptors such as Atg40 in yeast or RETREG1 in mammals have also been shown to form receptor complexes. Though, speculative at the moment, these receptor complexes could be involved in selective cargo recruitment and the crosstalk with other quality control pathways.Unlike most other reticulophagy receptors, C53 is not activated by nutrient starvation. Upon carbon- or nitrogen-starvation conditions C53 remains diffuse in the cytosol. This protein is only activated upon ER stress induction such as ribosome stalling. For example, a model poly-lysine stalling substrate that is targeted to the ER (ER-K20) induces C53 puncta formation. Similarly, chemicals that inhibit translational elongation, thereby leading to ribosome collisions are potent inducers of C53-dependent autophagy. Upon activation, C53 mediates the degradation of stalled nascent chains that are otherwise toxic to the cell. Quantitative proteomics analyses indicate rather than structural ER proteins, translocon components or ribosomes, C53 cargo clientele include cell wall and extracellular proteins that fold and mature at the ER.In our recent study , we idenOur current working model is that ribosome stalling or collisions induces ufmylation of RPL26, a 60S ribosomal protein . Once UF


Using a ground-based model to simulate spaceflight , an in-depth survey of the immune and hematological systems of mice at 7-days post-exposure was performed. Collected blood was profiled with a hematology analyzer and spleens were analyzed by whole transcriptome shotgun sequencing (RNA-sequencing). The results revealed negligible differences in immune differentials. However, hematological system analyses of whole blood indicated large disparities in red blood cell differentials and morphology, suggestive of anemia. Murine Reactome networks indicated majority of spleen cells displayed differentially expressed genes (DEG) involved in signal transduction, metabolism, cell cycle, chromatin organization, and DNA repair. Although immune differentials were not changed, DEG analysis of the spleen revealed expression profiles associated with inflammation and dysregulated immune function persist to 1-week post-simulated spaceflight. Additionally, specific regulation pathways associated with human blood disease gene orthologs, such as blood pressure regulation, transforming growth factor-β receptor signaling, and B cell differentiation were noted. Collectively, this study revealed differential immune and hematological outcomes 1-week post-simulated spaceflight conditions, suggesting recovery from spaceflight is an unremitting process. While the majority of spaceflight studies focus on the physiological responses during or immediately after flight, readaptation to nominal gravitational environments is also an important factor in astronaut health. This is particularly important for future exploratory missions to the Moon (0.166 g) or Mars (0.38 g) involving long-duration exposures to microgravity (0 g) and elevated doses of ionizing irradiation. Both the adaptation to spaceflight, and the readaptation to planetary gravitational environments, involve multiple physiological systems. Currently, short and the long-term consequences of prolonged exposure to deep spaceflight environments are largely unknown.1, myelin degeneration2, disorientation and motor control deficits4, visual deficits5, endocrine and hormonal alterations6, muscle tone wasting7, and bone loss due to lack of mechanical loading8. Upon return to Earth, there is a period of readaptation that includes acute motion sickness, unsteadiness, imbalance due to gravity-induced vestibular fluid shifts9, and blood pressure changes12. Most notably, space anemia14 was re-classified as post-flight anemia, because of inconsistencies reported in key hematological parameters, including red blood cell (RBC) counts and mean corpuscular volumes (MPV). This was attributed to the timing of blood sample collections occurring at post-flight return versus in-flight retrieval15. Mission duration can also affect post-flight recovery, whereby longer missions result in prolonged recovery post-mission16. This becomes an important risk factor to consider for exploratory missions to Mars. Indeed, understanding how immune and hematological cells compensate in response to the changing gravitational environment becomes particularly important as crew currently rehabilitate to Earth’s gravity for multiple months post-flight17. Yet, this timeframe may be problematic on planetary bodies with reduced gravity. Therefore, a better understanding of blood differentials and their transcriptional regulatory patterns are essential for successful development of countermeasures to be used on future exploratory missions to the lunar surface and Mars.There are several consequences to living in low-Earth orbit. These include edema due to fluid and cardiovascular deconditioning22. Here, we expand on this work, using RNA-sequencing of splenic tissues and complete blood cell (CBC) counts to characterize relevant immune and hematological pathways engaged after 1-week readaptation following simulated spaceflight conditions.Ground-based models of spaceflight are developed to easily explore the impacts of the spaceflight environment on physiology. Indeed, we previously reported that a 21-day, protracted exposure of 0.04 Gy low dose/low dose rate gamma radiation (LDR), combined with hindlimb unloading (HLU), can have neurological impacts on blood–brain barrier integrity, the oxidative stress response, and neuroplasticityPrincipal component analysis (PCA) plots were generated with each cohort Fig. A. Venn dp-value of 0.05, statistically significant Log2 fold change of DEG (simSpace versus control) were plotted23  or leukocytes, reticulated platelet and reticulocyte RBC, acting as a filter for circulating blood cells. Using NASA GeneLab’s volcano plot visualization tool with a maximum adjusted Slc genes involved in transporter signaling of sodium/potassium and cation elimination. Metabolism-related genes involved in liposome and glycoprotein formation were also highly upregulated, including Igf2r, B4galt2, and B4galt3 and the ubiquitin-pathways, including Ubap1, Fbxl22, and Ube2d2b. Cell cycle genes were also induced, including DEG associated with mitosis, such as Sssca1 and Kif24 and cytokinesis, such as Myh10 and Tor1aip1. Induction of DNA damage repair genes were also noted including, recombination repair genes, Swsap1 and Ippk and downregulation of DNA double stranded break repair gene, Ppp4r2.Murine Reactome pathway analysis was performed, which displayed pathways involved in signal transduction, metabolism, cell cycle, DNA repair, and chromatin organization at 7-days post-simSpace exposure Fig. . The majBlood was collected 7-days post-simulated spaceflight and WBC were counted with an automated analyzer, which displayed no significant differences between control and simSpace for immune differentials including, platelets Fig. A, mean pBcl11a, Nedd4l, and Aspn had common pathways involved in regulation of blood pressure, protein modifications, transforming growth factor-β signaling, anatomical structure regulation, and negative regulation of developmental processes  in post-simSpace cohorts, compared to controls Fig. A,B. Signses Fig. I.Figure 18. Analyses at 7-days post-simSpace provide insight into the immediate early responses similarly experienced by crew during Earth readaptation, a critical time point of equalizing homeostasis. Hematological profiling revealed minimal disparities in WBC differentials, however RBC displayed a wide variation in morphology and disrupted function, including reduced overall numbers and hemoglobin levels, suggestive of anemia. Transcriptomic analysis of the spleen, which is the filter for circulating blood cells and a secondary lymphoid organ, displayed genes involved in signal transduction, metabolism, cell cycle, chromatin organization and DNA repair pathways are altered. These analyses also revealed pathways connected to dysfunctional immunity and inflammation, including Cd19, Alox5, Trem1, and H2-DMb2 expression. Genes also implicated in blood disorders, including Bcl11a, Nedd4l, and Aspn, with common pathways involved in regulation of blood pressure, protein modifications, transforming growth factor regulation, and negative regulation of developmental pathways were identified. Thus, understanding immediate early immune and hematological pathways engaged post-simulated spaceflight sheds light on suitable timepoints for therapeutic intervention to promote homeostasis and restoration.Recovery post-spaceflight is systemically taxing and often leaves a lasting impression on human health. Circulating RBC and WBC interact with all physiological systems; therefore, a systems biology approach was applied to assess the effects of the immune and hematological outcomes post-simulated spaceflight (simSpace). Since astronaut collections are difficult to acquire, ground-based animal models are a useful alternative that provides valuable insight into these outcomes. Therefore, spleens and blood of mice were analyzed by RNAseq and hematological analyses, respectively, at 7-days post-simSpace. Previous studies in our lab have developed a simSpace model of low-dose radiation  and hindlimb unloading (HLU) that mimics astronauts in-orbit on the International Space Station (ISS)18, indicating LDR, HLU, and simSpace independently trigger divergent pathways post-exposure. Interestingly, post-HLU appears to produce a large number of transcriptional changes compared to post-LDR alone, indicating altered gravity may cause pronounced physiological impairments. However, different doses and types of irradiation experienced on deep space missions would also cause distinct results24; therefore, future studies are aimed to delineate these transcriptional profiles as well. In addition, combined exposures of post-simSpace (HLU + LDR) compared to single exposures of post-HLU or post-LDR alone also created increased expression profile variability, which may account for reduced DEG observed in post-simSpace  were observed, supporting either minimal physiological effects occurred during simSpace or recovery post-simSpace has reached homeostasis.DEG from spleens collected at 7-days post-simSpace were compared to single exposures of post-LDR or -HLU alone, which showed negligible overlap in genes Fig. A–C, indiSlc22a4. In fact, multiple Slc genes were upregulated post-simSpace, which are transporters of small molecules, including sodium/potassium and cation elimination, suggesting systemic electrolyte imbalance and potential dehydration. Of note, these ion channels are also mechanosensitive and regulate signaling pathways via physical forces they receive25. Therefore, more studies are required to better understand the contribution of these mechanosensitive transporter signaling pathways during- and post-simSpace.Reactome analysis identified five major functional pathways that were affected, including metabolism, cell cycle, chromatin organization, DNA repair, and signal transduction  Fig. . The maj28, while metabolic syndromes and ageing are also linked to oxidative stress29. In this study, multiple genes involved in metabolism were upregulated, including the insulin signaling receptor, Igf2r. Additionally, glycoprotein forming genes, B4galt2 and B4galt3 were both upregulated, suggesting heightened glycoprotein synthesis. Cell cycle pathways were also altered, including upregulation of genes Sccca1, Kif24, Myh10, and Tor1aip1. These DEG are involved in mitosis and meiosis via microtubule formation and cytokinesis, suggesting increased cellular turnover and division post-simSpace. Chromatin organization and remodeling genes Chd3 and Ncoa3 downregulation were also identified. Furthermore, Swsap1, Ift57, and Ippk genes were upregulated, indicating DNA repair mechanisms occur post-simSpace. Collectively, multiple DEG pathways were actively engaged at 7-days post-simSpace, indicating a dynamic and constant healing process seeking homeostasis.We and others have identified the oxidative stress response as a major pathway engaged during spaceflightCard11, Sf3b1, H2-DMb2, Vps39, Ccdc50, and Pcm1; while SphK1, Trem1, and Alox5 were upregulated  suggests phagocytosis impairment31. In line with this, impairment of neutrophil phagocytosis is observed up to 3-days post-spaceflight33, suggesting a possible role for downregulated Vps39 in this process. The transcription factor NFκB activates a wide variety of signaling pathways involved in immune activation, inflammation, and cytokine production34. Card11 and Ccdc50 are both regulators of NFκB signaling. Their expression levels are downregulated, indicating dysregulated immunity. In addition, Alox5 is typically upregulated by the proinflammatory cytokine, GM-CSF, involved in the generation of emergency myelopoiesis and activates matured myeloid cells into a pro-inflammatory state35. Therefore, Alox5 upregulation suggests a proinflammatory environment predominates post-simSpace. In line with this, Trem1 is upregulated and is involved in amplifying inflammation of neutrophils and monocytes36. Indeed, Trem1 inhibition has been successfully shown to prevent mir-155-driven lung inflammation and injury37, septic shock38, and neuroinflammation39. Trem1 is expressed in neutrophils, which are granulocytes40. Elevated granulocytes and granulocyte-to-lymphocyte ratio (GLR) has been identified as a biomarker for inflight immune monitoring41, therefore, it is plausible that Trem1 induction may also be a determinant for inflight inflammation. Acute inflammation is beneficial in the face of immunological challenges, such as infections or tumors, yet chronic persistence, specifically post-exposures can be detrimental to the host. Resolution of inflammation does not appear to occur at 7-days post-simSpace, suggesting the potential disease development42. Furthermore, although correlation of Alox5 and Trem1 expression levels and severity of inflammation is not clear, monitoring Alox5 and Trem1 upregulation may be useful biomarkers to measure chronic inflammation in future spaceflight studies.Immune-related genes were grouped according to their cellular function. Myeloid cell (monocyte and granulocyte)-related genes included downregulation of ted Fig. D. H2-DMbSla, Cd19, Pikap1, Kbtbd8, Pcm1, Card11, and Cccdc50; and upregulation of Sphk1 and Zfp341 , suggesting the importance of regulating this biomarker in particular for future spaceflight studies.CBC indicated no differences in WBC counts, which may be due to either group-housing effects on cell distributionels Fig. A,B. ThesHCT Fig. D,E. HoweHCT Fig. C,F,G. Co54. Using Malacards Human Disease Database, outstanding blood disease/disorder orthologs including, upregulated Tmem184a, Daam2, Ippk, Aspn, RhD, and Igf2r; and downregulated Nedd4l and Bcl11a were identified  was noted post-simSpace, which may have been induced due to RBC loss, as Rh(null) phenotypes are associated with hemolytic anemia55. As it is unclear if anemia was induced during- or post-simSpace in our model, and due to the complex definition of space/post-flight anemia, more studies are required. Nonetheless, while determining the timeframe of anemia onset is important, we propose the potential for prolonged and pronounced anemia manifestation in astronauts on partial gravity surfaces of the Moon and Mars, following long duration, deep space travel. Therefore, identifying biomarkers to assist with future countermeasure developments to help safeguard astronauts on future deep space missions is critical. Interestingly, pathway analysis implicated Bcl11a, Aspn, and Nedd4l are interconnected and regulate a wide variety of pathways including, blood pressure, B cell differentiation, and TGF-βR signaling  were acclimatized in standard habitats at 20 °C with a 12 h:12 h light:dark cycle for 7 days. Following acclimatization, animals were housed one per cage and assigned to one of four groups: (1) control (n = 6); (2) hindlimb unloaded (HLU) (n = 3); (3) low-dose irradiated  (n = 6); and (4) simSpace, combination of hindlimb unloaded and low-dose irradiated (HLU + LDR) (n = 6) for 21 days. Low-dose/low-dose rate was delivered using 57Co plates  placed 7 cm below the cages, with 1 plate per 2 cages for whole-body irradiation. Uniformity of dose was ± 5%, as previously described22. Post-exposure animals were group housed (3 per cage) for 7-days. Commercial pellet chow  and hydrogel were available ad libitum. Health status, water and food intake were daily monitored. After 7-day post-simSpace, mice were CO2 euthanized and spleens were collected and frozen. RNA was isolated from whole, frozen spleens and samples were subjected to whole transcriptome shotgun sequencing (RNA-sequencing)18. Datasets were deposited to the NASA GeneLab Data Systems (GLDS)-211 and were analyzed using the NASA pipeline for determination of differentially expressed genes (DEG)23. All methods were performed in accordance with the guidelines recommended in the Guide for the Care and Use of Laboratory Animals57 and was approved by the Institutional Animal Care and Use Committee (IACUC) at Loma Linda University (Protocol number 8130028). This study is reported in accordance with ARRIVE guidelines58.Six-month-old, female 2 at day seven after the simulated spaceflight period of 21-days. Whole blood was collected via cardiac punch in [K2]-ethylenediaminetetraacetic acid (EDTA) coated syringes immediately following euthanasia and evaluated using the ABC Vet Hematology Analyzer . Parameters characterized include red blood cell (RBC), platelet (PLT), and white blood cell (WBC) counts, hemoglobin concentration (HGB) and hematocrit . Mean corpuscular volume , mean corpuscular hemoglobin , mean corpuscular hemoglobin concentration , RBC distribution width , and the mean platelet volume  were also reported, along with absolute counts of granulocytes, monocytes, and lymphocytes (× 106 cells/ml).Mice were euthanized with 100% CO2 euthanized followed by immediate exsanguination by cardiac puncture.Mice were humanely COSpleens were isolated and placed in a sterile cryovial, snap frozen in liquid nitrogen and kept at − 80 °C. Spleens were homogenized with CKMix  and a Minilys homogenizer (Bertin Instruments). AllPrep DNA/RNA/miRNA Universal Kit  extracted RNA and DNA according to the manufacturer’s instructions. RNA and DNA concentrations were measured using a Qubit 3.0 Fluorometer  and stored at − 80 °C.23.To construct RNA-sequencing libraries, an Ovation Mouse RNA-Seq System 1–96  was used per manufacturer’s instructions. RNA integrity was determined using a 2100 Bioanalyzer (Agilent) with RIN values > 8. 100 ng of total RNA was used as input. cDNA (first and second strands) was synthesized from total RNA spiked with ERCC ExFold RNA Spike-In Mix 1  at the appropriate ratio. Products were sheared using Covaris S220 Focused-ultrasonicator  to obtain fragment sizes between 150–200 bp. Followed by end-repair, adaptor index ligation and strand selection. For multiplexing, barcodes with unique indices out of 96, were used per sample. Custom InDA-C primer mixture SS5 Version5 for mice (NuGEN Technologies) was used for strand selection. Libraries were amplified by PCR (17 cycles) on a Mastercycler Pro (Eppendorf) and purified with RNAClean XP Agencourt beads . Libraries were sequenced on a HiSeq 4000  to generate 15–30 M 75-bp single end reads per sample. Raw data are available at NASA GeneLab 59 using the Trim Galore! wrapper, sequencing reads were trimmed. All reads were mapped with STAR60 and expressions were quantified and imported with RSEM61 and tximport62 respectively, into DESeq2 (R Bioconductor)63. Log2 fold-change (0.263) and adjusted p-value cutoffs (0.05) were used. All groups were compared using the Wald test and the likelihood ratio test was used to generate the F statistic p-value. Reactome version 6764 was used to detect enriched murine pathways. Volcano plots were generated using NASA’s GeneLab tool23. Murine spleen DEG that were orthologous to human blood gene-related diseases, as defined by GeneCards Human Gene Database and MalaCards Human Disease Databases, were identified. Using the Network Analyst Global EnrichNetwork tool, with parameters including the gene ontology: biological process (GO:BP) enrichment analysis database, large graph layout, and single node display , the blood gene-related gene list was mapped to determine common pathways.With Cutadaptt test with Welch’s correction was performed for all CBC parameters. A GitHub data analysis notebook was created and can be found here, https://github.com/jgalazka/GLDS-211_Paul_2021.Differential expression analysis was performed in R DESeq2. All groups were compared using the Wald test and the likelihood ratio test was used to generate the F statistic p-value. A one-way ANOVA with post-hoc (Sidak) test was performed for the calculated coefficient of gene expression variation per gene. An unpaired, parametric Supplementary Information 1.Supplementary Information 2.


The SARS-CoV-2 variant of concern (VOC) “Delta” is currently defined by PANGOLIN as a cluster of 33 different AY sublineages. Delta (in particular B.1.617.2) is largely and rapidly replacing the Alpha VOC as the dominant clade in most countries. To date, variations in the Spike protein of the Delta VOC have largely been limited. We report here the results of a genomic surveillance programme from Northern Italy. We identified several Delta sublineages harbouring mutations previously reported in GISAID at extremely low frequencies and in different combinations. Two patients (one of them vaccinated) tested positive for a Delta sublineage harbouring S71F, T250I, T572I and K854N. More patients tested positive for G769 V plus C1248F, A352S, and R158G and C1248F, respectively. Genomic surveillance of Delta variants should be encouraged to anticipate immune escape and deploy countermeasures. SARS-CoV-2 is an RNA virus with a relatively stable genome, largely thanks to its proofreading exonuclease. Nevertheless, the aggressive pandemic has led to massive replication events worldwide, and around 800 SARS-CoV-2 sublineages have been reported to date. The most challenging sublineages  have been named “variants of concern” (VOC). The WHO has recently dubbed VOCs with a simplified Greek alphabet nomenclature, easy to recall than former phylogenies provided by GISAID, PANGOLIN, NextStrain, and Public Health England (PHE). While in the first semester of 2020 Alpha (a.k.a. B.1.1.7 or “UK variant”) was the predominant VOC, the second semester of 2021, after massive vaccination campaigns, saw the growth of Delta (a.k.a. “Indian variant”). The Delta VOC, identified in October 2020 in India, consists of 41 different sublineages sharing additional T19R, del157/158, T478K, and D950N in Spike protein and I82T in M protein compared to B.1. The beta-loop-beta receptor-binding motif (RBM) adopts an altered but stable conformation causing separation in some of the antibody binding epitopes [According to Italian Ministry of Health guidelines, we started a genomic surveillance programme in the Lombardy Region in Northern Italy.https://outbreak.info/situation-reports).Between June 2021 and July 2021, 2,622 (8.8%) out of 29,666 nasopharyngeal swabs (NPS) tested positive for SARS-CoV-2 RNA at our laboratory by using several real-time reverse transcriptase (RT)-PCR platforms. 815 of these 2,622 positive samples underwent sequencing for characterizing the infecting SARS CoV-2 variant, according to the surveillance programme of the Italian National Institute of Health and Ministry of Health. Of these 815, 97 samples were sequenced by Sanger for the entire Spike gene or by next-generation whole-genome sequencing (NGS) as previously reported . BrieflyOut of the 97 Spike or full-genome sequences, 76 (78%) harboured the Delta variant. Among them, we describe here 5 patients who harboured sequences with additional Spike mutations very rarely reported (or unreported in such combination) in the GISAID database. A first sublineage was identified in two patients, a 65-years old male and a 26-years old female nurse. The male patient was an international trader and had a history of 2 former NPS positivities, the first in Italy in November 2020 and the second in Kenya (where he had moved since April 2021) in May 2021, both during voluntary screenings while asymptomatic. After a 15-days quarantine in Kenya, he fled to South Africa, where he fell ill with severe pneumonia and tested positive for a third time on June 7, 2021. He was repatriated with a dedicated flight and admitted to the hospital on June 15, 2021. His anti-S IgG serologies  were negative on February 2021 and on June 17. The patient fully recovered, his NPS was negative on June 25 and was discharged the following day. The female patient was a nurse at the COVID hospital who was quarantined before having contact with the male trader. She had been fully vaccinated with BNT162b2 since January 2021, and follow-up anti-S IgG serologies (DiaSorin) were positive on March 1 (367 AU/ml) and June 18 (752 AU/ml). On June 10 she had short contact with her father, who had tested positive for a SARS-CoV-2 antigen assay the day before and had got the infection from a Tunisian immigrant: she fell ill on June 13 and tested positive at PCR on June 14. Her 20-years old brother, who lived with the father, fell ill on June 10, tested positive on June 14, and developed severe pneumonia: none of the nurse colleagues tested positive at surveillance NPSs. The patient recovered and a follow-up NPS on July 1 was negative. Both the trader and the nurse harboured identical B.1.617.2 sequences, additionally harbouring S71F, T250I, T572I, and K854N [A second sublineage was identified in a 44-years old immunocompetent female whose NPS tested positive (cycle thresholds 22 for gene E and 23 for gene N2) on June 22, after strict contact with her mother, which was a confirmed COVID-19 case admitted to the hospital two days before. The patient had received 2 doses of Vaxzevria® on March 15 and June 1, 2021. She developed anosmia, ageusia, mild fever, fatigue, and dyspnoea. The Spike sequence harboured G769 V and C1248F as additional mutations. These mutations were previously unreported in Delta VOCs.A third sublineage was reported in a 23 years-old male who tested positive on July 13 (cycle threshold 18 for gene E and N2). The sequence confirmed Delta variant additionally harbouring A352S.A fourth sublineage was reported in an unvaccinated, immunocompetent 45 years-old male returning from Barcelona by plane on June 1. He developed myalgia and mild fever, and tested positive to NPS on June 4 and 9; a follow-up NPS was negative on June 16. His 74-years old immunocompetent father (who had received a single Vaxzevria® dose) tested positive on June 12, developed mild respiratory symptoms, and tested negative on June 23. His 84-years old immunocompetent mother (who had received 2 doses of Comirnaty® on March 31 and April 21), first tested positive on July 5. The sequence confirmed Delta variant additionally harbouring R158G and C1248F.The Delta VOC is becoming the predominant SARS-CoV-2 strain worldwide, largely replacing the Alpha (B.1.1.7) VOC thanks to higher transmissivity.Panthera leo persica) in Arignar Anna Zoological Park, Chennai, Tamil Nadu, India [The differences between the main Delta sublineages are summarized in u, India . Analysiu, India . Neutralu, India .Figure AY.2 additionally harbours V70F (unique among delta lineages), A222V  and K417N (as for AY.1) K356N and V1228L have also been rarely reported. AY.3 additionally harbours E156G. AY.3.1 never harbours T95I nor A222V, and harbours F157C and R158G at about 10% frequencies.As expected for a strain with massive circulation, quasi-species are arising across the world. Subclades for Alpha and other VOCs have been relatively uncommon , but Delta moves from as a mix of 41 sublineages, making further diversification easier. Our analysis derives several limitations from GISAID: deposited sequences are a sample of the total number of cases and may not represent the true prevalence of the mutations in the population, and case numbers for any given lineage/mutation can be significantly affected by overall case numbers and rates of genomic sequencing at any given location.in vitro neutralization by convalescent plasma from previous waves [We have reported here the heterogeneity of the Delta VOCs in a small series from a single Italian laboratory, which suggests the real heterogeneity of Delta is far higher than previously supposed. The Delta VOC has reduced to us waves , 13–16, us waves , 14, 17,us waves  and vaccus waves , mRNA-12us waves , 22–24, us waves , 26, Ad2us waves , CoronaVus waves , 28 and us waves . Given s


BACH2 and BCL6 RNA had a shorter overall survival (OS). Their low RNA expression was associated with a shorter overall survival of immunoglobulin heavy chain variable region-mutated (IGHV-M) CLL patients, as well as those with 11q and 13q deletions. Further, co-immunoprecipitation and mass spectrometry studies from MEC-1 CLL cells showed no direct interaction between BACH2 and BCL6, but shared protein networks that are involved in ubiquitination mediated B-cell receptor functions, nucleic acid metabolism, protein degradation, and homeostasis in CLL biology. Taken together, this study reports BACH2 as a potential prognosticator and indicates the protein networks influenced by BCL6 and BACH2 in CLL.Chronic lymphocytic leukaemia (CLL) is the most frequent type of leukaemia with a highly variable presentation, clinical course, and outcome. The overall aim of our study was to determine the clinical and functional significance of two B-cell regulators, BACH2 and BCL6, in CLL. The results showed that CLL patients expressing low levels of BCL6 and BACH2 RNA had significantly shorter overall survival (OS) than high BCL6- and BACH2-expressing cases. Notably, their low expression specifically decreased the OS of immunoglobulin heavy chain variable region-mutated (IGHV-M) CLL patients, as well as those with 11q and 13q deletions. Similar to the RNA data, a low BACH2 protein expression was associated with a significantly shorter OS than a high expression. There was no direct interaction observed between BACH2 and BCL6 in MEC-1 CLL cells, but they shared protein networks that included fifty different proteins. Interestingly, a prognostic index (PI) model that we generated, using integrative risk score values of BACH2 RNA expression, age, and 17p deletion status, predicted patient outcomes in our cohort. Taken together, these data have shown for the first time a possible prognostic role for BACH2 in CLL and have revealed protein interaction networks shared by BCL6 and BACH2, indicating a significant role for BACH2 and BCL6 in key cellular processes, including ubiquitination mediated B-cell receptor functions, nucleic acid metabolism, protein degradation, and homeostasis in CLL biology.Chronic lymphocytic leukaemia (CLL) is a heterogeneous disease with a highly variable clinical outcome. There are well-established CLL prognostic biomarkers that have transformed treatment and improved the understanding of CLL biology. Here, we have studied the clinical significance of two crucial B cell regulators, BACH2  and BCL6 (B-cell CLL/lymphoma 6), in a cohort of 102 CLL patients and determined the protein interaction networks that they participate in using MEC-1 CLL cells. We observed that CLL patients expressing low levels of  In cont B-cells . These c B-cells . In contreaction ,6. DespiBcl6−/+ Bach2−/+ mice exhibit a profound reduction in GC formation in response to T-cell dependent antigen immunization [BACH2 was studied in untreated CLL patients with age-matched healthy donors, BACH2 mRNA expression was shown to be reduced in CD4+ T-cells, CD8+ T-cells, and leukemic B-cells [The transcriptional repressor, BACH2, is required for the somatic hypermutation (SHM) and class switch recombination (CSR) of antibody genes, GC formation, and the inhibition of plasma cell (PC) differentiation in B-cells ,8,9. BACnization . In GC Bnization . Interacnization . Interesnization . In diffnization  and BACHnization . Moreovenization . Recentl B-cells . Here, wThis retrospective study included 102 CLL patients following informed consent. Patients were selected based on the availability of fresh samples, clinical and cytogenetic data, as well as overall survival data up to June 2015. This study cohort is heterogeneous, reflecting the diversity typically found among CLL patients. Samples were obtained through the Newcastle Biobank (17/NE/0361). All patients were diagnosed with CLL according to World Health Organization (WHO) criteria. RNA and protein extraction, qRT-PCR, Western blot, and statistical analysis from patient samples were performed using standard techniques, as detailed in the BACH2 and BCL6 were measured in a cohort of 102 and 91 among 102 CLL patients  than those expressing high levels  (CLL expressing low levels of the ctively) . In relactively) A, whereactively) . These fctively) , it inclBACH2 RNA had a significantly shorter OS than those expressing high levels in the treatment naïve cohort, indicating BACH2 as a potential prognosticator , but survival analysis showed that a low RNA expression of BACH2 and BCL6 was related to a poor outcome in the 13q-   , which is in CLL .We identified a positive correlation between BCL6 and BACH2 expression , suggestAmong the 50 identified interaction partners, 28 proteins were localised in the cytoplasm, while 7 were in the cell periphery/membrane . A further 15 of the interactors display catalytic activity, of which 13 are enzymes that catalyse metabolic interconversion  .Several components of the eukaryotic 26S proteasome complex that are part of the two subcomplexes—the 20S core particle (CP) and the 19S regulatory particle —are idenThe second largest group of proteins that interact with BACH2 and BCL6 belong to nucleic acid metabolism . Mini-chromosome maintenance proteins (MCMs) are a family of six structurally related helicases (MCM2–7) that play critical roles in DNA replication and genome stability by forming a variety of complexes ,33. EachIn the same theme of ubiquitination, genome stability, and DNA damage, TCOF1 (treacle ribosome biogenesis factor 1) has also been reported to function in ubiquitination through E3 ubiquitin ligase complex, RNA biogenesis, mitosis, proliferation, DNA damage response, and apoptosis ,41. TherTP53 [BACH2 and BCL6 predicts a shorter OS in IGHV-M, 13q-deleted (regarded as a known low-risk subgroup), and 11q-deleted subgroups. In addition, low BACH2 RNA and protein expression also predicted a shorter OS in treatment naïve CLL patients. The number of treated and treatment naïve patients were fairly equally distributed and balanced within most of the subgroups used in the univariate and multivariate analyses (The precise cellular origin of CLL remains unclear and the current knowledge of CLL biology demonstrates no direct link between the proliferative circulating CLL cells to a specific normal B-cell subset ,47. B-ceTP53 ,52. Hereanalyses . Howeveranalyses  were tooThe deletion of 13q14 is the most frequent genetic lesion in CLL, being most prevalent in IGHV-M CLL ,55. It iBCL6 RNA and protein expression, as well as its mutational status in CLL, have been investigated over the years with conflicting reports [The prognostic role of  reports ,62,63, p reports ,63,64,65 reports ,67, as t reports ,69. This reports ,67,68,69Our co-immunoprecipitation studies, performed in MEC1 CLL cells, show no direct interaction between BACH2 and BCL6, but the majority of the interaction partners found in the shared protein interaction network suggested cooperation between them in key signalling processes, such as the ubiquitin-mediated protein degradation pathway, nucleic acid metabolism, and cellular homeostasis . FurtherBACH2 as a possible independent, integrative predictor of outcome in CLL. The clinical outcome and the proteomic data highlight the tumour suppressor role and the functional significance of BACH2 and BCL6 in CLL biology. Further investigations that study the impact of the BACH2 and BCL6 signalling axis will not only improve our understanding of CLL pathogenesis, but will also offer new opportunities for novel targeted therapy development.Taken together, our data for the first time demonstrate


Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease. During the past decade, novel pathogenic mechanisms of IPF have been elucidated that have shifted the concept of IPF from an inflammatory-driven to an epithelial-driven disease. Dysregulated repair responses induced by recurrent epithelial cell damage and excessive extracellular matrix accumulation result in pulmonary fibrosis. Although there is currently no curative therapy for IPF, two medications, pirfenidone and nintedanib, have been introduced based on understanding the pathogenesis of the disease. In this review, we discuss advances in understanding IPF pathogenesis, highlighting epithelial–mesenchymal transition (EMT), the ubiquitin-proteasome system, and endothelial cells. TGF-β is a central regulator involved in EMT and pulmonary fibrosis. HECT-, RING finger-, and U-box-type E3 ubiquitin ligases regulate TGF-β-Smad pathway-mediated EMT via the ubiquitin-proteasome pathway. p27 degradation mediated by the SCF-type E3 ligase, Skp2, contributes to the progression of pulmonary fibrosis by promotion of either mesenchymal fibroblast proliferation, EMT, or both. In addition to fibroblasts as key effector cells in myofibroblast differentiation and extracellular matrix deposition, endothelial cells also play a role in the processes of IPF. Endothelial cells can transform into myofibroblasts; therefore, endothelial–mesenchymal transition can be another source of myofibroblasts. Interstitial lung disease (ILD) is a diverse group of respiratory diseases with varying pathology. ILD and pulmonary fibrosis, an advanced pathological condition, are characterized by lung parenchyma injury with various patterns of interstitial inflammation, cell proliferation, and fibrosis within the alveolar wall and interstitium ,2,3. TheIdiopathic pulmonary fibrosis (IPF) is the most common form of idiopathic interstitial pneumonia and idiopathic fibrotic lung disorder. IPF occurs primarily in middle-aged and older adults, typically in the sixth and seventh decades, and its incidence increases remarkably with age ,5,6. IncFibrosis results in impaired ventilation, functional respiratory impairment, reduced gas exchange, and respiratory failure . IPF is The diagnostic criteria for idiopathic interstitial pneumonias and IPF have changed several times ,5,12, anThere is currently no curative medication for IPF . In pastThere are numerous studies demonstrating that inflammation can be quite pronounced in IPF pathogenesis and, formerly, inflammation was regarded as the main cause of fibrosis ,8,14. AnA variety of profibrotic mediators, such as TGF-β, PDGF, and connective tissue growth factor (CTGF), and their signaling cascades play important roles in the pathogenesis of fibrotic diseases ,23. DuriTGF-β is a dimeric polypeptide growth factor. TGF-β regulates cell proliferation and differentiation, immune regulation, embryonic development, and angiogenesis ,26. In tTGF-β is critical in the initiation and progression of all types of tissue fibrosis  27] and and27] aThe TGF-β system is complex and interacts with other cell-signaling pathways. TGF-β promotes the production of profibrotic mediators and other proangiogenic mediators. In addition to TGF-β, a variety of other secreted factors, including tumor necrosis factor-α (TNF-α) , CTGF 2, PDGF, aRegarding interleukins, levels of the proinflammatory cytokine, IL-18, are elevated in serum, airway epithelium, and alveolar macrophages of patients with IPF . HoshinoIn fibrotic diseases, unidentified triggers alter inflammatory and fibrotic responses. However, these triggers are not specific and the development and progression of IPF depend on interactions with environmental factors and genetic disease susceptibility ,23. RepeTERT), desmoplakin (DSP), and A-kinase anchoring protein 13 (AKAP13) [MUC5B) is found in pulmonary fibrosis patients [MUC5B overexpression in airway epithelial cells. This minor allele was present at a frequency of 38% in patients with IPF. The odds ratios for disease among patients who were heterozygous and those who were homozygous for the minor allele of this SNP were 9.0 (95% CI: 6.2–13.1) and 21.8 (95% CI: 5.1–93.5), respectively [Age-related changes, a genetic susceptibility, epigenetic factors, and mitochondrial dysfunction determine disease susceptibility ,41. Epig(AKAP13) . A commopatients ,14,20,38patients . The minectively . Howeverectively . Wolter Studies using animal models enable the pathogenesis of diseases to be interrogated and facilitate preclinical assessment of drug candidates ,23. For Fibroblasts from patients with IPF exhibit an abnormally “activated” phenotype and have global alterations in DNA methylation, which may contribute to fibroblast heterogeneity among patients with IPF . In IPF,Fibroblastic foci are regions of highly proliferative myofibroblast accumulation that are located immediately adjacent to regions of hyperplastic or apoptotic epithelial cells . MyofibrThere has been impressive progress in understanding the pathogenesis of IPF. In the later part of this review, we focus on advances regarding the ubiquitin-proteasome system and endothelial cells involved in the development of pulmonary fibrosis.Morphologically, pulmonary endothelial cells are contiguous to epithelial and mesenchymal cells and form the endothelial lining of blood vessels . The end123I-metaiodobenzylguanidine washout from the lungs [+ cell population obtained was cultured as mouse pulmonary endothelial cells. In endothelial cells from bleomycin-treated lungs, the expression levels of endothelial injury markers, PAI-1, von Willebrand factor, and matrix metalloproteinase-12, were elevated at the early stage and sustained after a few weeks, which suggested that endothelial cells were damaged in the intratracheal bleomycin-induced pulmonary fibrosis.Although pulmonary endothelial cell injury has not been considered a main clinical topic in patients with ILDs, some clinical studies have focused on endothelial cell damage. Takabatake et al. showed pulmonary microvascular endothelial injury during a fibrotic process using the rate of he lungs . Magro ehe lungs . Endothehe lungs . Based ohe lungs . Mouse p2, maintain respiratory homeostasis by modulating platelet aggregation, inhibiting leukocyte adhesion, and controlling vascular smooth muscle cell proliferation [2 in response to agonists. Basically, endothelial cells constitutively synthesize and release intracellular nitric oxide and prostaglandin I2 in response to agonists [2 in response to thapsigargin stimulation in endothelial cells was investigated during the process of bleomycin-induced lung injury. Thapsigargin is a selective inhibitor of the endoplasmic reticulum Ca2+-ATPase and increases intracellular Ca2+ levels through plasma membrane store-operated Ca2+ channels. In normal conditions, the addition of thapsigargin increases the production of nitric oxide and prostaglandin I2. The response to the addition of thapsigargin in endothelial cells from intratracheally bleomycin-induced pulmonary fibrosis mice was maintained, but the levels of intracellular nitric oxide were reduced in endothelial cells from bleomycin-treated mice. The concentration of prostaglandin I2 was estimated based on that of its stable metabolite, 6-keto PGF1α. Thapsigargin simulation increased 6-keto PGF1α production in endothelial cells isolated from bleomycin-treated mice. However, the production of 6-keto PGF1α in response to thapsigargin was lower in cells isolated from bleomycin-treated mice, which showed that endothelial cell function based on thapsigargin reactivity was attenuated in endothelial cells from bleomycin-treated mice at the fibrotic phase.Endothelium-derived relaxing factors, intracellular nitric oxide, and prostaglandin Iferation ,57. Althferation , althougagonists . The proNitric oxide synthase is affected by inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) expression. iNOS is stimulated by inflammation. eNOS overexpression reduced lung collagen accumulation, histological changes, and mortality in bleomycin-induced pulmonary fibrosis mice . On the Epithelial–mesenchymal transition (EMT) is a pathophysiological process in which epithelial cells lose some of their epithelial characteristics and gain mesenchymal ones. This molecular programming occurs in three biological contexts, development, cancer, and fibrosis, in which tissue injury and remodeling disrupt normal tissue homeostasis . During 1α in endothelial cells. Both bleomycin exposure and TGF-β substantially changed endothelial cells, functionally and phenotypically.TGF-β is a central regulator of myofibroblast recruitment, activation, and differentiation during tissue repair  and is oTGF-β signaling promotes induction of EMT and expression of fibrosis associated genes and participates in progression of tissue fibrosis, such as kidney and lung fibroses . TGF-β bUbiquitin-mediated protein degradation plays crucial roles in controlling the amount of cellular proteins and participates in various cellular processes such as development, cell proliferation, differentiation, signal transduction, and apoptosis. Deregulation of the system is caused by various diseases, including cancers, neurodegenerative diseases, and tissue fibrosis. E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin ligase) coordinately promote polyubiquitin conjugation to target proteins. Polyubiquitylated proteins are recognized and proteolytically degraded by the proteasome . E3 is rSmurf (Smad ubiquitination regulatory factor) HECT-type E3 ligases, such as Smurf1/2, act as an accelerator to enhance TGF-β signaling by promoting ubiquitin-dependent degradation of Smad7 . Smurf2 NEDD4L (NEDD4-2) is a membrane bound HECT-type E3 ligase that targets various membrane proteins including ENaC and TβR . Cell tyHECT-type E3 ligases, such as Smurfs and NEDD4L, and a single RING-type E3 Arkadia participate in modulation of fibrosis via regulation of the TGF-β-Smad pathway . ElkouriU-box-type E3 ligases, such as STUB1 and CHIP, participate in ubiquitin-mediated degradation of Smads in TGF-β-signaling. NOX4 promotes TGF-β-mediated myoblast differentiation by stimulation of reactive oxygen species (ROS) induction. STUB1 targets Smad3  and NOX4Reports have shown that TRB3 participates in regulation of TGF-β signaling. TRB3 is a mammalian Tribble-related protein that is involved in various cellular processes as a scaffold or adaptor to promote degradation of target proteins such as Smurf1/2 and to regulate multiple signal transduction pathways such as TGF-β and MAPK signaling . In mousRecently, small molecule inhibitors of HSP90 (Heat shock protein 90) have attracted attention as therapeutic drugs for IPF ,98. HSP9Marinova et al. reported that HSP90 inhibitor, AUY-922, suppressed lung injury and fibrosis in a hydrochloric acid-induced lung injury model . SolopovKip1 (p27) and p21Cip1 (p21), participate in regulation of the growth and differentiation of both epithelial and interstitial cells in various tissues [EMT of alveolar epithelial cells to α-SMA-positive myofibroblasts and promotion of fibroblast proliferation participate in development of pulmonary fibrosis. In cell proliferation, cyclin-dependent kinases (CDKs) play important roles as the cell cycle engine. CDK inhibitors (CKIs) such as p27 tissues . A compl tissues ,109. SCF tissues ,110. The tissues .In the progressive kidney obstruction mouse model, Skp2 deficiency suppresses renal fibrosis and the progression of renal fibrosis is partially recovered by Skp2/p27-double deficiency ,113. In Similarly, in the bleomycin-induced pulmonary fibrosis model, accumulation of both collagen A1 and fibronectin and the score of pulmonary fibrosis are reduced by Skp2-deficiency . MoreoveSCF-Fbw7 targets several growth promoting proteins such as c-Myc, c-Myb, Notch, and GATA2/3, whereas SCF-Skp2 targets several anti-proliferate proteins such as p27 and p21 . Wang etIPF is a progressive and fatal disease with 2.5–3.5 years being the median survival time after diagnosis. Although incomplete, understanding of IPF has recently progressed. The criteria for IPF diagnosis were recently revised to emphasize the significance of the radiological UIP pattern and multidisciplinary discussion between pulmonologists, radiologists, and pathologists in the diagnostic process. Although there is currently no curative treatment for IPF, promising medications, including pirfenidone and nintedanib, have been introduced. They reduce the decline in lung function in patients with IPF, but have a small impact on survival and quality of life.Advances in understanding fibrogenic processes are likely to yield more effective therapies that give longer survival and better quality of life. The current concepts of IPF pathogenesis focus on epithelial cell damage, the TGF-β system, and epithelial–mesenchymal transition. It would be helpful to consider other processes in the pathogenesis of pulmonary fibrosis, such as the ubiquitin-proteasome pathway and endothelial cells. Many targeted therapies are currently in early-phase clinical trials. Signaling related to the ubiquitin-proteasome pathway may be another therapeutic target. Further studies with multifaceted and broader approaches will contribute to better understanding of the molecular pathogenesis of pulmonary fibrosis.


Taken together, our study identified that MMP12 might be a useful tumor biomarker and therapeutic target for ESCC.Neo-chemoradiotherapy (nCRT) before surgery is a standard treatment for locally advanced esophageal cancers. However, the treatment outcome of nCRT varied with different patients. This study aimed to identify potential biomarkers for prediction of nCRT-response in patients with esophageal squamous cell carcinoma (ESCC). Microarray datasets of nCRT responder and non-responder samples (access number GSE45670 and GSE59974) of patients with ESCC were downloaded from Gene Expression Omnibus (GEO) database. The mRNA expression profiles of cancer biopsies from four ESCC patients were analyzed before and after nCRT. Differentially expressed genes (DEGs) and miRNAs were screened between nCRT responder and non-responder ESCC samples. Functional enrichment analysis was conducted for these DEGs followed by construction of protein-protein interaction (PPI) network and miRNA-mRNA regulatory network. Finally, univariate survival analysis was performed to identify candidate biomarkers with prognostic values in ESCC. We identified numerous DEGs and differentially expressed miRNAs from nCRT responder group. GO and KEGG analysis showed that the dysregulated genes were mainly involved in biological processes and pathways, including “response to stimulus”, “cellular response to organic substance”, “regulation of signal transduction”, “AGE-RAGE signaling pathway in diabetic complications”, and “steroid hormone biosynthesis”. After integration of PPI network and miRNA-mRNA network analysis, we found eight genes, TNF, AKR1C1, AKR1C2, ICAM1, GPR68, GNB4, SERPINE1 and MMP12, could be candidate genes associated with disease progression. Univariate cox regression analysis showed that there was no significant correlation between dysregulated miRNAs  and overall survival of ESCC patients. Moreover, abnormal expression of MMP12 was significantly correlated with pathological degree, TNM stage, lymph nodes metastasis, and overall survival of ESCC patients ( Esophageal squamous cell carcinoma (ESCC) is a primary histological type of esophageal cancer worldwide. In the United States, approximately 18,440 new cases were diagnosed and 13,100 deaths occurred in 2020 . China iEsophageal cancer has a poor prognosis when it is diagnosed at advanced stage. The 5 years survival rate would decrease to 4% when metastasis occurred . As for In recent years, the development of microarray analysis and RNA sequencing technology provided favorable information for exploring the association of gene expression and clinical outcomes . In thisThe gene expression profile GSE45670  and miRNhttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE137867) were obtained by RNA sequencing technology and mRNA expression was analyzed for these clinical specimens. GSE137867 were considered as validation datasets. DEGs were screened from the three datasets and overlapped genes were considered as candidate genes related to ESCC.In addition, cancer biopsies were taken from four patients before and after nCRT in Changzhou No.2 People’s Hospital, the Affiliated Hospital of Nanjing Medical University. This study was approved by the Ethical Research Committee of hospital. Transcriptomics data .PCA is a multivariate technique that converted multiple correlated variables into cohort of values linearly, and uncorrelated variables represented principal components. It has been extensively used to explore high-dimensional data, such as genomic and transcriptomic expression data . In thisp < 0.1 and fold change >1.5 (|log2FC| > 0.585). Moreover, with the same cutoff criteria, we screened the overlapped DEGs of GSE137867 and GEO datasets to conduct further analysis.The miRNA matrix and mRNA expression profiles were normalized based on quantile normalization method. Limma package  was usedp < 0.05 was considered as significant difference, and the column with smaller p value represented a closer association between DEGs and pathway categories.Gene Ontology (GO)  and KyotWe further predicted the miRNA-mRNA interactions by examining various databases [TargetScan , miRTarBhttps://string-db.org/) (The online tool STRING (db.org/)  was useddb.org/)  was usedIn addition, we integrated the PPI network and miRNA-mRNA regulatory network by using Cytoscape software to predict the candidate miRNAs involved in ESCC progression.Survival package  in R sofp < 0.1 and fold change >1.5 (|log2FC| > 0.585), we screened a total of 1311 DEGs from nCRT responders and non-responders. We found 672 upregulated and 639 down-regulated genes. The same cutoff criteria were applied for RNA-Seq profile GSE137867, and numerous DEGs were screened in specimens before and after nCRT.PCA analysis and correlation analysis on samples were conducted for mRNA expression dataset GSE45670. Under the threshold of P < 0.1 and fold change >1.5 (|log2FC| > 0.585). Scatter plot and volcano plot were used to visualize the results. Finally, we identified 44 upregulated miRNAs and 15 downregulated miRNAs from the nCRT responders compared with non-responders. The differentially expressed miRNAs are listed in Moreover, we integrated the overlapped DEGs between RNA-Seq profile and GEO datasets. Of these gene overlaps, 17 genes were both upregulated in two datasets while eight genes were both downregulated in two datasets . Volcanop = 2.85e−03).The GO analysis results revealed that these dysregulated DEGs were mainly associated with several biological processes , such asBy predicting the target genes of the upregulated miRNAs and downregulated miRNAs, we built two miRNA-target networks for upregulated miRNAs  and downUsing the online tool STRING and Cytoscape software, we constructed a PPI network for DEGs  and obtaBy integrating PPI network and miRNA-mRNA network, we finally constructed a regulatory network that consisted with miRNAs and genes. Among these genes, PER2, PCTP, TNF and hsa-miR-486-5p exhibited higher interactions than other genes. Thus, these genes were identified as hub genes related to ESCC progression.p < 0.05).We conducted a survival analysis on independent cohort of patients (the data were derived from TCGA database) to analyze the prognostic significance of 8 genes in ESCC patients. The analyzed signatures included genes TNF, AKR1C1, AKR1C2, and ICAM1, et al.  The patiMoreover, univariate cox regression analysis and survival analysis were performed for these differentially expressed miRNAs, such as hsa-miR-34b-3p, hsa-miR-127-5p, hsa-miR-144-3p, and hsa-miR-486-5p et al. However, the results revealed that there was no significant correlation between any dysregulated miRNAs and prognosis of ESCC patients .In this study, we attempted to identify potential tumor biomarkers for prediction of nCRT-response in patients with ESCC. After integrating overlapped genes between RNA sequencing dataset and GEO datasets, we identified 17 genes that were upregulated, and eight genes that were downregulated in these two datasets. After verification of the data set samples used in the project analysis and the actual number of samples included in the analysis, no samples were excluded from the project, and all samples were analyzed. Functional enrichment analysis showed that dysregulated genes were mainly involved in “response to stimulus”, “regulation of signal transduction”, “AGE-RAGE signaling pathway in diabetic complications”, “steroid hormone biosynthesis”. According to analysis of PPI network and miRNA-mRNA network, we identified eight candidate genes, TNF, AKR1C1, AKR1C2, ICAM1, GPR68, GNB4, SERPINE1 and MMP12, that were associated with progression of ESCC progression. Finally, univariate regression analysis in ESCC patients revealed that high expression of MMP12 was significantly correlated to poor prognosis.Previous studies have explored the difference of gene expression between nCRT responder and non-responder samples for prediction of nCRT response in esophageal cancer. Our results demonstrated that eight genes, including TNF, AKR1C1, AKR1C2, ICAM1, GPR68, GNB4, SERPINE1 and MMP12, were candidate genes associated with ESCC progression. AKR1C1/C2 encodes enzymes belong to aldo/keto reductase superfamily. These enzymes are crucial in drug resistance of cancer cells and related to metabolism of polycyclic aromatic hydrocarbons . AKR1C1 In the miRNA-mRNA regulatory network, we identified PER2, PCTP, TNF and hsa-miR-486-5p as hub genes related to disease progression. MiRNA have been confirmed to have a major role in cancer development by regulating the expression of oncogenes or cancer suppressor genes. Aberrant expression of special miRNAs have been detected in ESCC patients such as upregulated of miR-10b , miR-21 In addition to hsa-miR-486-5p, survival analysis was performed for several other differentially expressed miRNAs, such as hsa-miR-34b-3p, hsa-miR-127-5p, and hsa-miR-144-3p, et al. in tumor samples. However, there was no significant correlation between any dysregulated miRNAs and prognosis of ESCC patients. According to literature, miR‐127 was downregulated in several types of cancers, including gastric cancer , glioblaProtein of MMP-12 is known as human macrophage metalloelastase (HME) or macrophage elastase (ME), and belongs to the MMPs family. MMP12 is associated with elastin degradation and macrophage migration in various diseases, such as chronic obstructive pulmonary disease, skin diseases and cancers . HoweverThere were some limitations in our study. Experimental validation should be conducted to identify the exact biological behaviors of candidate DEGs or miRNAs in ESCC development. Meanwhile, the number of ESCC specimen was limited. Further validation in larger cohorts is necessary to investigate the disease predictive value of these genes.In conclusion, we identified eight genes, including TNF, AKR1C1, AKR1C2, ICAM1, GPR68, GNB4, SERPINE1, and MMP12, as candidate genes by performing integrative analysis on gene expression profiles of microarray datasets. We found that abnormal expression of MMP12 was significantly correlated with pathological degree, TNM stage, lymph nodes metastasis, survival time of ESCC patients. Further basic experiments and large-scale multi-center clinical research studies are required to validate our results since our study was conducted based on data analysis.


Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest types of cancer, and it is currently the third most common cause of cancer death in the U.S.A. Progress in the fight against PDAC has been hampered by an inability to detect it early in the overwhelming majority of patients, and also by the reduced oxygen levels and nutrient perfusion caused by new matrix formation through the activation of stromal cells in the context of desmoplasia. One harbinger of PDAC is excess intrapancreatic fat deposition, namely, fatty pancreas, which specifically affects the tumor macro- and microenvironment in the organ. Over half of PDAC patients have diabetes mellitus (DM) at the time of diagnosis, and fatty pancreas is associated with subsequent DM development. Moreover, there is a strong association between fatty pancreas and fatty liver through obesity, and a higher intrapancreatic fat percentage has been noted in acute pancreatitis patients with DM than in those without DM. All these findings suggest that the link between fatty pancreas and PDAC might occur through metabolic alterations, either DM-related or non-DM-related. Based on clinical, in vivo and in vitro evidence, the current review highlights the etiologies of fatty pancreas (including fatty infiltration and replacement) and the fatty pancreas-associated metabolic alterations involved in oncogenesis to provide crucial targets to prevent, detect, and/or effectively treat PDAC. Approximately 90% of these cancers are pancreatic ductal adenocarcinoma (PDAC) , among tectively . Moreoveectively . In addiectively . It has ectively ; these lectively , and oneectively  and PanIectively . Nearly ectively ; a stronectively ,13. Indeectively . Taken tSeveral protocols are used to measure pancreatic fat via magnetic resonance imaging (MRI), with MR spectroscopy (MRS) being considered equivalent to histology. Although histology remains the gold standard for assessing pancreatic fat content, routine biopsy sampling to evaluate fatty pancreas is not feasible .With regard to histology, there is currently no validated objective scoring system available for the histologic assessment of fatty pancreas . Based oFor MRI, there are several methods to measure fatty pancreas , includiHypotheses to explain the association between obesity and pancreatic cancer risk include hormonal and inflammatory effects of adipose tissue, increased exposure to carcinogens, reduced physical activity, obesity-induced hypoxia resulting in increases in vascular endothelial growth factor  and an aG12D (KC) or the EWSR1-FLI1 oncogene (E/F) in pancreas cells exhibited prominent acinar cell mass depletion and extensive lipomatosis. Moreover, E/F mice exhibit spontaneous ADM formation without the development of neoplastic lesions [Changes in cellular identity within the pancreas can be triggered by recurrent attacks of pancreatitis and low-grade inflammation. These changes may lead to pancreatic carcinogenesis , and aci lesions . Postn i lesions .ob) and obese hyperleptinemic (Lepdb) mice has a higher total pancreatic fat, triglyceride and free fatty acid content but lower phospholipid and cholesterol content. Triglycerides and free fatty acids represent the toxic components of adipose tissue, and both phospholipids and cholesterol are integral constituents of cell membranes; thus, despite an overall increase in adiposity, the membrane stability and fluidity appear to remain constant in the case of fatty pancreas [Compared to lean mice, the pancreas of leptin deficient (Leppancreas .In mice exposed to high-fat diets (HFDs), the pancreas seems to be more susceptible to fat deposition than the liver; additionally, higher insulin resistance, increased pancreatic fat, increased inflammation, greater fibrosis , larger The islets of non-obese cotton rats fed a normal diet increase in size through β-cell hyperplasia with insulin resistance, and adipocytes accumulate in the pancreas but not in the liver during aging .Gestational DM in rats causes long-term effects on the pancreas of offspring, whereas n-3 polyunsaturated fatty acids have a beneficial role . This isIn animal models, excess intrapancreatic fat is a driver of pancreatic carcinogenesis . For exaThroughout life, there is important interplay between the endocrine and exocrine pancreas . Below, Three mechanisms have been proposed to induce fatty pancreas-associated exocrine pancreatic disorder: the lipotoxicity of acinar cells, adipocyte-mediated negative paracrine effect, and direct destruction of acinar cells . A study2 and serum triglycerides ≥ 1.7 mmol/l and/or T2DM [2 and serum triglycerides ≥ 1.7 mmol/l and/or T2DM, pancreatic fat is positively associated with only two metabolites: lysine-derivate and the bile acid conjugate taurodeoxycholate [Overall, it remains debated whether fatty pancreas is associated with glucose metabolism, including DM, insulin resistance, β-cell function, prediabetes, fasting glucose, glucose tolerance, glycated hemoglobin, T2DM risk, metabolic syndrome and a longitudinal decrease in endogenous insulin-secreting capacity. For example, positive associations were noted in a meta-analysis of 13 studies involving 49,329 subjects , a meta-/or T2DM ; 106 sub/or T2DM ; 14 T2DM/or T2DM ; 132 con/or T2DM ; 143 pat/or T2DM ; 79 pati/or T2DM ; 50 age-/or T2DM ; 685 hea/or T2DM ; 250 con/or T2DM ; and 320/or T2DM , 112 vol/or T2DM , 64 pati/or T2DM , 51 subj/or T2DM , 45 subj/or T2DM , and 78 /or T2DM  without /or T2DM ; a rando/or T2DM , and stu/or T2DM , 73 pers/or T2DM , 158 chi/or T2DM , 8 monoz/or T2DM , 86 pati/or T2DM , pancrea/or T2DM , 1367 vo/or T2DM , 56 part/or T2DM , and 97 /or T2DM . Theoret/or T2DM , apoptos/or T2DM , all of /or T2DM ,104,105./or T2DM ,108, and/or T2DM . Notably/or T2DM ,81, T2DM/or T2DM , central/or T2DM  and lipo/or T2DM  are fact/or T2DM . In patiycholate . In patiFollowing acute pancreatitis, levels of triglycerides , leptin The arbitrary threshold for considering fatty liver as a histology feature is the presence of lipid droplets in >5% of hepatocytes . The pheThe links between fatty pancreas and PanIN , IPMN 44,131, eaReprogrammed metabolism and metabolic crosstalk within the tumor microenvironment contribute to unlimited pancreatic tumor proliferation . An earlThe reprogrammed metabolism of PDAC is summarized below.Otto Warburg demonstrated that tumor cells consume more glucose than normal cells. Tumor cells subsequently convert most glucose-derived carbon into lactate, even in the presence of sufficient oxygen, a process known as the Warburg effect or aerobic glycolysis . In PDACIn contrast to normal cells relying on dietary fat, >90% of triacylglycerol fatty acids in tumor cells are synthesized de novo . Thus, iAutophagy and macropinocytosis are critical nutrient supply pathways that support the high rate of proliferation for pancreatic tumor growth in nutrient-deprived environments . AutophaPDAC has the highest case fatality rate of any solid tumor, highlighting the urgency for novel therapeutic strategies to combat this deadly disease. Currently, targeted or immunotherapy strategies effective for most patients with PDAC are lacking. Accumulating animal and human evidence suggests that fatty pancreas is involved in PDAC development. Both fatty infiltration and fatty replacement lead to fatty pancreas and may predispose toward the development of PDAC in different or complementary ways. Further investigations to clarify the crucial factors that cause fatty pancreas, particularly those associated with metabolic oncogenesis, are needed. Such research is vital for discovering effective therapeutic strategies to combat PDAC by preventing the development of fatty pancreas.