Long-term glucocorticoids can alter sperm motility, vitality, or morphology, disrupting male reproductive function. This study scrutinized the synergistic benefits of two Egyptian plants against dexamethasone (Dexa)-induced testicular and autophagy dysfunction in male rats. Phytochemical ingredients and the combination index were estimated for Purslane ethanolic extract (PEE) and Chicory water extract (CWE). Four control groups received saline and 100 mg/kg of each PEE, CWE, and PEE/CWE, daily for 8 weeks. Dexa (1 mg/kg daily for 6 weeks) induced infertility where PEE, CWE, and PEE/CWE were given. Seminal analysis, male hormones, glycemic and oxidative stress markers, endoplasmic reticulum (ER) stress markers (Sigma 1R and GRP78), and autophagy regulators  were measured. The in vitro study illustrated the synergistic (CI < 1) antioxidant capacity of the PEE/CWE combination. Dexa exerts testicular damage by inducing oxidative reactions, a marked reduction in serum testosterone, TSH and LH levels, insulin resistance, ER stress, and autophagy. In contrast, the PEE and CWE extracts improve fertility hormones, sperm motility, and testicular histological alterations through attenuating oxidative stress and autophagy, with a synergistic effect upon combination. In conclusion, the administration of PEE/CWE has promised ameliorative impacts on male infertility and can delay disease progression. Infertility and problems of impaired fecundity have been considered the most serious social problems facing developed and industrialized countries. Infertility has been defined as the failure to achieve conception after 12 months or more of regular unprotected sexual intercourse . Of all Oxidative stress is among the core causes of male infertility ,18. DuriFurthermore, ER stress and oxidative stress play significant roles in the pathophysiology of male infertility by inducing autophagy in testes, causing testicular degeneration and dysfunction ,23. AutoPortulaca oleracea L.) and Chicory (Cichorium intybus L.) are edible Egyptian plants that belong to the family Portulacaceae and Asteraceae, respectively [P. oleracea leaves are a rich source of dietary antioxidants, including flavonoids , glutathione, omega-3 fatty acids, alkaloids, and vitamins, as well as dietary minerals [According to previous assumptions, the Mediterranean diet and different natural remedies rich in flavonoids and antioxidants can promote the elimination of pollutants and hinder oxidative stress and testicular damage through different mechanisms ,29,30,31ectively ,33,34. Pminerals . Phytochminerals . The secminerals ,38. The minerals ,40. BothBased on these premises, the present study was designed to investigate the synergetic protective effects of a combination of Purslane and Chicory extracts against Dexa-induced testicular damages by studying the changes in the testicular morphology, spermatogenic indices, and biochemical parameters in adult rats. Collectively, we elaborate the role of Dexa-induced oxidative stress in the aggravation of ER stress and autophagy signaling via exploring the levels of the glycemic markers, ER stress regulators (Sigma 1R and glucose-regulated protein 78 (GRP78)), and autophagy regulators responsible for autophagosome initiations (P-mTOR(S2448), beclin-1, phosphatidylinositol 3-kinase class III (PI3KC3)), formation (autophagy genes ATG5 and ATG7), and maturation  in the rat’s testis.2O2, Tris-HCl, 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB), reduced glutathione (GSH), Griess reagent (Sulphanilamide and N-1-naphthyl ethylenediamine), sulfosalicylic acid, and dexamethasone (Dexa) were obtained from Sigma-Aldrich . ELISA kits were purchased from COBAS, USA. Other assay kits were purchased from Spectrum, Egypt. Easy redTM total RNA extraction kit, cDNA synthesis kit, and 2X SYBR green master mix kit were obtained from iNtRON Biotechnology, Korea. Primers were purchased from Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA. The following antibodies were used: Anti-beclin-1 , phosphoinositide-3-kinase class 3 , Anti-Sigma R1 polyclonal antibody , Phospho-mTOR (Ser2448) , Anti-P62/SQSTM1 (D1Q5S) rabbit mAb , LC3B antibody-N-terminal , glucose-regulated protein 78 (GRP78) polyclonal antibody , and β-actin . Other chemicals were obtained with high grades.Folin–Ciocalteau reagent, aluminum chloride, nitroblue tetrazolium (NBT), 1-1-diphenyl 2-picrylhydrazyl (DPPH), phenazine methosulfate (PMS), nicotinamide adenine dinucleotide reduced (NADH), gallic acid, catechin, thiobarbituric acid, trichloroacetic acid (TCA), Cumene HPortulaca oleracea L. (Purslane) leaves and Cichorium intybus L. (Chicory) roots (y) roots  were colFresh samples were collected, cleaned with water, and were split as roots, stems, and leaves. Leaves were crushed into powder by a plant grinder after being dried by sun exposure at room temperature . The obtApparently, healthy Chicory roots were collected, washed thoroughly in tap water, dried at room temperature, and then lyophilized at −80 °C to be powdered. Dried powdered root (500 g) was soaked in 500 mL water for 4 days. The extract was decanted, filtered through cheesecloth, and was reduced to 10% of its original volume , then lyThe total phenolic content of PEE and CWE was determined using the Folin–Ciocalteau colorimetric method [Total flavonoid content was quantified calorimetrically using the aluminum chloride method, and catechin was used as standard [c method . The resstandard . Data arThe mineral content  of PEE or CWE was assessed. One gram of PEE and CWE was digested in a hydrochloric acid/nitric acid mixture (3:1) for 1 h at 100 °C. The digested extracts were proceeded to be available for the analysis using atomic absorption spectroscopy  according to the US EPA Method 200.7 [od 200.7 ,45 and Uod 200.7 . Data arVitamins content: Folic acid, vitamin C, and vitamin A were analyzed in the Laboratory Unit for Advanced Environmental and Biological Analysis, High Institute of Public Health, Alexandria University. Folic acid was extracted from PEE and CWE according to the method described in AOAC [w/v ratio) according to the modified method of Abushita, Hebshi [ in AOAC . The HPL, Hebshi . The mix, Hebshi . As a fa, Hebshi .3PO4, methanol, and acetonitrile. The device was equipped with a variable-wavelength detector set at 284 nm. Retention time and peak spectra of the standard phenolic compounds—ran at the same chromatographic conditions—were used for identification. All phenolic compounds were expressed as µg/g extract.Phytochemical components of PEE and CWE were identified and quantified by an Agilent 1260 Infinity HPLC series , as described by Lu, Yuan . HPLC anFor the antioxidant assays, the extracts were dissolved in dis. water (10 mg/mL) and concentration-dependent dilutions were made. The radical scavenging activity of the extracts was evaluated using 1-1-diphenyl 2-picrylhydrazyl (DPPH), nitric oxide (NO), and superoxide radical scavenging methods, as well as total antioxidant capacity.DPPH scavenging activity: According to Manzocco, Anese [o, Anese , PEE ando, Anese .The superoxide anion scavenging activity: The superoxide anion radicals were generated by NADH oxidation and assayed by a reduction in NBT. The reaction mixture (3.0 mL) contained 1.0 mL extract (of different concentrations), 0.5 mL of NBT (0.3 mM), 0.5 mL NADH (0.936 mM) and 0.5 mL Tris–HCl buffer . The reaction was started by the addition of 0.5 mL PMS solution (0.12 mM) to the reaction mixture and incubated at 25 °C for 5 min. The absorbance was measured at 560 nm against a blank sample [k sample .Nitric oxide scavenging activity: According to Marcocci, Maguire [ Maguire , 100 µL sample is the absorbance of the extracts and Acontrol is the absorbance of the control (with no extract).The percentage of the radicals scavenging was calculated as follows:IC50 values were calculated to determine the 50% inhibition of the radicals. IC50 of both PEE and CWE for each assay was estimated, and the values were used for the mixture (IC50 PEE/IC50 CWE). Different ratios were used  to estimate the antioxidant activities. A 1:1 (IC50 PEE: IC50 CWE) ratio showed the most synergetic effect; so, it was used in the in vivo study for the combined extracts.Total antioxidant capacity was carried out using the phosphomolybdenum method [m method . An aliqSixty-four sexually mature Sprague-Dawley male rats, weighing about 165–180 g (10–12 weeks), were purchased from the animal house of the Institute of Graduate Studies and Research, Alexandria University, Egypt. All rats were raised in polypropylene cages  and fed on a standard diet with ad libitum tap water. They were kept on a 12:12 h light-dark cycle in a well-aerated room under a controlled environment. All animal procedures were accepted under the ethical standards of scientific research of the Institutional Animal Care and Use Committee (IACUC), Alexandria University .After one week of acclimatization, rats were divided into eight groups  and treated for 8 weeks. There are four control groups that received saline (Sham), PEE (100 mg/kg), CWE (100 mg/kg), and PEE/CWE combined (100 mg/kg), daily for eight weeks. Infertility-induced group: received dexamethasone  daily for six weeks. There are three treated groups that received: PEE , CWE , and PEE/CWE combined . The animals of the treated groups were continuously administered their respective treatments via gavage daily for two weeks before Dexa and continued for another six weeks with Dexa. Based on earlier studies, a daily dose of 100 mg/kg of either PEE or CWE was chosen ,57,58.At the end of the experimental period, the rats were fasted overnight and sacrificed by decapitation. Blood samples were collected from the inferior vena cava. Sera were separated by centrifugation and stored at −20 °C for glycemic markers and hormone measurements.w/v). The supernatant was kept at −80 °C until used for enzymatic and non-enzymatic analysis .Testicles from each animal were dissected. For histopathological examination, four right testes/group were fixed in 10% neutral formalin. The other four right testes/group were homogenized for the seminal analysis assessment. The left testis was kept at −80 °C for biochemical and molecular measurements. The testicular tissue was crushed in Tris HCl buffer   at 37 °C. Testicular sperm count, morphology index, and motility were assessed manually using a hemocytometer. All procedures were carried out at 37 °C, and everything that came in contact with sperm was pre-warmed and kept at 37 °C. Each sample was counted four times and averaged .Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone levels were evaluated using ELISA kits  according to the manufacturer’s instructions. The absorbance was measured at 450 nm , and their levels were expressed as μIU/mL (FSH and LH) and ng/dl (testosterone).Serum and testicular fructose levels (sperm energy source) were determined according to Foreman, Mongar [, Mongar . A totalTesticular α-mannosidase (EC 3.2.1.24) activity was assayed as described by Tulsiani, Skudlarek [d-mannopyranoside (4 mM) and 20 µL of the testis homogenate supernatant were added to 100 µL of sodium acetate buffer, pH 4.4, and incubated for 60 min at 37 °C. The reaction was stopped by adding 100 µL of alkaline buffer . The p-nitrophenol released was quantized by measuring the absorbance of the sample against the blank at 400 nm. One unit of the PNP-α-mannosidase activity is the enzyme that catalyzes the release of 1 µmol p-nitrophenol per hour. The α-mannosidase activity was expressed as U/mg protein.kudlarek . BrieflySerum insulin and fasting blood glucose (FBG) levels were measured using a commercial kit obtained from COBAS, Germany, and Spectrum-diagnostics, Egypt, respectively. Insulin sensitivity was estimated by homeostasis model assessment (HOMA-IR), and the β-cell function was evaluated by HOMA-β according to the following equations :HOMA−IR=malondialdehyde (MDA), one of the aldehyde products of lipid peroxidation. MDA reacts with thiobarbituric acid under acidic conditions forming a pink-colored product on heating, measured spectrophotometrically at 532 nm [Lipid peroxidation was assessed calorimetrically in testicular tissues by measuring the formation of t 532 nm . The tesThe testicular reduced glutathione (GSH) level was measured in the clear supernatant using Ellman’s method. After protein removal, GSH interacts with Ellman reagent , generating a yellow-colored 2-nitro-5-thiobenzoic acid product. The reaction between GSH and DTNB was analyzed at 412 nm. The GSH level was expressed as mM/mg protein.testicular glutathione peroxidase (GPx) (EC 1.11.1.9) was determined based on the reduction of cumene hydroperoxide via GSH oxidation into glutathione disulfide (GSSG) [The activity of e (GSSG) . The amoThe testicular glutathione-S-transferase (GST) (EC 2.5.1.18) activity was spectrophotometrically determined using 4-nitrobenzyl chloride (pH 6.5) and GSH as substrates forming glutathione nitrobenzyl [robenzyl . The absTotal testicular proteins were determined using Lowry method, where BSA (1 mg/mL) was used as a standard [standard . Total pThe protein levels of testicular Sigma 1 receptor (Sigma 1R), phosphatidylinositol 3-kinase Class III (PI3KC3), and Beclin-1 were measured by the quantitative ELISA technique using rabbit polyclonal sigma 1R , PI3KC3  and Beclin-1 . The antigen was diluted in a coating buffer  to a final concentration of 100 µg protein. In duplicate, samples were loaded in microtiter plate wells and incubated for 2 h at room temperature and overnight at 4 °C. A blocking solution of 5% BSA was utilized and incubated for 1 h at room temperature. To decrease the non-specific binding, primary and secondary antibodies were diluted in a blocking solution (2%). The primary antibodies were incubated for 1 h at room temperature before being kept at 4 °C overnight. The plate was washed six times with washing buffer before being incubated for 2 h at room temperature with alkaline phosphatase-conjugated secondary antibody . ρ-Nitrophenyl phosphate disodium salt was used as ALP substrate and allowed for 15 min before the addition of sodium hydroxide (3 M) was used to stop the reaction. The developed color was measured at 450 nm on a plate reader. Their levels were expressed in ng/mg protein using the proteins’ standard curves.TM total RNA extraction kit  and measured spectrophotometrically. Then, 1 µg of total RNA was reverse transcribed using Maxime RT PreMix kit  following the manufacturer’s protocol. The qRT-PCR analysis was performed as follows: 1 µL of cDNA was added to appropriate primers (10 µM), 10 µL of RealMOD™ Green w2 2X qPCR mix , and RNase-free water in a final volume of 20 µL per well. Quantitative PCR was performed in Roto Gene Q 5Plex HRM , applying the following thermal protocol: 95 °C for 2 min, followed by 35–40 cycles of 95 °C for 20 s, 43–60 °C for 30 s, and 72 °C for 60 s. Samples were loaded in duplicate. The comparative 2−ΔΔCT method was used to calculate the data, and all values were normalized to the GAPDH gene’s mRNA level. The results are expressed as an n-fold increase in gene expression using the calibrator results of the sham control. Total RNA from the testis was extracted according to the manufacturer’s instructions using an easy redTesticular tissue was homogenized in ice-cold Nonidet-P40 (NP-40) buffer with the addition of 1% protease inhibitor and phosphatase inhibitor cocktail. The lysates supernatant was separated by cool centrifugation at 13,000 rpm for 20 min, and their protein content was measured spectrophotometrically, followed by boiling in 2× Laemmli buffer for protein denaturation. Equal pooled protein concentrations (40μg) were loaded into 12% SDS-PAGE for separation and transferred onto nitrocellulose membranes . Membranes were blocked in 1× Tris-buffered saline/0.1% Tween 20 (TBST) with 5% BSA for 1 h at room temperature and incubated with the primary antibody specific to LC3B , Phospho-mTOR(Ser2448) , P62/SQSTM1 , GRP78 polyclonal antibody  and β-actin  for 1 h at room temperature then overnight at 4 °C. The membrane was washed six times (10 min each) in 1× TBST and incubated with goat anti-rabbit alkaline phosphatase-conjugated secondary antibody for 2 h at room temperature. Then nitro blue tetrazolium/5-Bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) solution  was added to visualize the protein bands. Bands were measured with Image Studio Lite software . All expressed proteins were normalized to the β-actin content, and their relative expression was calculated as the fold change of control.The nature of the combined effects of PEE and CWE was determined using the method described by Zhou, Li . The comWhile for other in vivo tested parameters,The CI was calculated using the formula:A CI of less than, equal to, or more than 1 indicates a synergic, additive, or antagonistic effect, respectively .Testicular tissues were removed from different rat groups after scarification. They were fixed in 10% neutral formalin and processed into paraffin blocks. Hematoxylin and eosin (H&E)-stained, four-microns-thick sections were mounted on glass slides . They wep < 0.05 regarded as statistically significant. SPSS 16.0  was utilized for these studies.The values were presented as the mean ± SD. Differences within groups were statistically examined using one-way ANOVA using the LSD test, with a The current results demonstrated that PEE and CWE contain various phytochemical constituents, minerals, and vitamins with different concentrations, as shown in Moreover, the minerals and vitamin analyses indicate that PEE and CWE are mineral- and vitamin-rich nutrients due to the presence of Na, K, Ca, Fe, Mg, and P in great amounts, with Cu, Mn, Zn, and Se in the least amount. PEE contains a greater amount of vitamin A, while CWE includes a higher amount of folic acid and vitamin C, p˂ 0.05) superior  than that of CWE, associated with a significant (p˂ 0.05) higher total antioxidant capacity than that of CWE. Concerning the synergistic analysis, the combination of PEE and CWE (PEE/CWE) showed a synergistic (CI ˂ 1) scavenging effect against DPPH, NO, and superoxide radicals . Although the Dexa-injected group showed a significant increase in the sperm count, this increase was associated with a significant decrease in the morphology index, sperm progressive motility, and % motility during the 1st, 2nd, and 3rd hours, with a significant increase in the non-progressive motility relative to values of the control group (p < 0.05). The groups injected with Dexa and supplemented with PEE, CWE, or their combination had a significant (p < 0.05) improved semen quality as compared to the Dexa-untreated group  decline in serum and seminal fructose levels and seminal α-mannosidase activity relative to the control group. In comparison, the three treated groups  illustrated a significant (p < 0.05) elevation in these markers relative to the Dexa group. Concerning the hormonal status, the Dexa-injected untreated group revealed a significant (p < 0.05) decrease in serum levels of male hormones  relative to the control group. On the other hand, administration of either PEE, CWE, or their combination to Dexa-injected rats resulted in a significant (p < 0.05) increase in FSH, LH, and testosterone levels relative to the Dexa group. The combination of PEE and CWE (Dexa+ PEE/CWE group) synergistically (CI < 1) elevated the α-mannosidase activity, FSH, LH, and testosterone levels . In contrast, a marked decrease in testicular MDA level and concurrent increases in GSH content and GPx and GST activities were observed in all treated rats. The combination of PEE and CWE revealed a synergistic (CI < 1) antioxidant effect, as indicated for all the studied oxidative stress parameters  reduced the FBG, insulin, and HOMA-IR levels relative to the Dexa-untreated group. Furthermore, the CWE and PEE/CWE treated groups showed a significant increase in the HOMA-β index compared to both the Dexa and PEE + Dexa groups. Moreover, the Dexa + PEE/CWE group reached levels comparable to the control group (The FBG and insulin levels in the Dexa-injected group displayed a significant increase accompanied by a significant elevation in HOMA-IR level and a significant reduction in HOMA-β level versus the control group (ol group . Interesol group . Therefop < 0.05) elevation in the protein levels of GRP78 and Sigma 1R was detected in the testicular tissues of Dexa-induced infertile rats relative to the control group (p < 0.05) attenuated the increased GRP78 and Sigma 1R protein levels relative to the Dexa-untreated group.Sigma 1R and GRP78 are important contributors to the ER stress response. A significant  elevated in Dexa-injected rats compared to the control rats. LC3II (p < 0.05) elevated in the Dexa-induced group compared to the control group. On the other hand, the quantitative RT-PCR analysis indicated a significant (p < 0.05) downregulation of the p62 mRNA expression level (p < 0.05) reduction in P62 protein level compared to the control group. The P-mTOR(S2448) protein level (p < 0.05) reduced in the Dexa-injected rats compared to the control rats. Together, these results indicated that the autophagy rates are enhanced in the Dexa-injected rats and that autophagy may be involved in the pathogenesis of male infertility.To investigate the involvement of Dexa and ER stress in autophagy modulation, the autophagosomal regulators, including PI3KC3, ATG5, ATG7, Beclin-1, LC3-II, P-mTOR(S2448), and P62, were detected at the protein and/or gene expression levels. Concerning the autophagy initiation and processing, the PI3KC3 protein level M and auts. LC3II E,F and Bs. LC3II I,J mRNA on level H in Dexaon level G confirmin level B was sigp < 0.05) ameliorating effect in these parameters compared to the Dexa-untreated group. The combined impact of the PEE/CWE combination on reducing the autophagy process was greater than the individual treatments with either PEE or CWE. P-mTOR(S2448), P62, Beclin-1, LC3-II, ATG5, and ATG7 revealed a synergistic effect (CI < 1), while PI3KC3 showed an additive effect (CI = 1). These outcomes suggest that combinations of PEE with CWE may synergize the inhibition of Dexa-induced male infertility through attenuation of ER stress and autophagy.The expression of autophagy-related proteins following PEE, CWE, or PEE/CWE treatment was evaluated to delineate their role in alleviating Dexa-induced testicular toxicology. Administration of PEE and CWE to the Dexa-injected rats attenuated the autophagy and introduced a significant  showed a better protective effect on Dexa-treated group . The SI Synthetic GCs are important long-acting steroid hormones used to treat everything from severe infection, asthma, to arthritis. Most patients are treated with low doses over a long period. However, long-term or high-dose usage of GCs can lead to a series of side effects . Dexa atSeveral studies have revealed that Dexa acts as a testicular toxicant. Dexa induces histopathological alterations and spermatogenesis defects, including epithelial vacuolizations, reduction in seminiferous tubule diameter, the elevation of the apoptotic index of germ cells, poor spermatogenesis, and significant maturation arrest ,59. ProlAccordingly, in the current study, Dexa injections for six constitutive weeks impaired spermatogenesis function and negatively affected the sperm morphology index and motility with a non-significant effect on the sperm count compared to the control group. Several studies indicated that the Dexa treatment decreases sperm motility ,59 due tThe detrimental effect of Dexa on the spermatogenesis index may also be due to the reduced fructose level and α-mannosidase activity. Fructose is the main sugar related to metabolism and sperm motility. It is an important energy source and a marker of the performance of seminal vesicles. Testosterone regulates seminal fructose levels by controlling the secretion function of the accessory glands ,86. As aIn the male reproductive system, ROS exhibits a dual role. At minimal physiological concentrations, ROS function as signaling molecules and exerts a vital physiological role in regulating the HPG axis, steroidogenesis, and spermatogenesis, as well as enhancing sperm capacitation, hyperactivation, and acrosome reactions. However, overproduction of ROS pushes the cellular redox balance towards an oxidative state linked to several male fertility problems, including varicocele, leukocytospermia, and idiopathic infertility ,89. Ther2O2 and lipid peroxides) at the expense of GSH. Furthermore, GSTs are important phase II detoxification enzymes that catalyze the conjugation of GSH with various electrophilic compounds [Previous studies showed that Dexa induces excessive ROS production and testicular oxidative stress in rodents ,84. Lipiompounds . In the ompounds , MukherjIncreased oxidative stress appears to be a probable signal for insulin resistance, which can be blocked by a wide range of antioxidants . In the In addition, the present study showed that autophagy was over-activated in the testis of Dexa-administered rats, implying that autophagy may play a role in Dexa-induced spermatogenesis deficiency. Autophagy, or programmed cell death type II, is an evolutionarily conserved process that involves the construction of a double-membrane vesicle called an autophagosome, fusion with lysosomes, and destruction of cytosolic components by resident hydrolases ,102,103.Concerning the autophagic process, ATG5, ATG7, and LC3 are involved in phagophore expansion and promote the formation of autophagosomes. ATG5 and ATG7 are two important autophagy-related proteins as they participate in the initiation of autophagosome formation. Overexpression of ATG5 or ATG7 can increase autophagic activity as a pro-survival mechanism to protect cells under stress conditions . LC3 is Herein, Dexa upregulated the autophagy-associated genes, including ATG5, ATG7, PI3KC3, Beclin-1, LC3II, and the LC3II/LC3I ratio, associated with downregulation of the P-mTOR(S2448) and P62 protein levels. Together, these results indicate that the autophagy rates are enhanced in Dexa-injected rats, and that autophagy hyperactivation may be involved in the pathogenesis of male infertility. Therefore, Dexa demonstrated a rat model of testicular damage through activation of testicular oxidative stress and insulin resistance that aggravate ER stress and autophagic degradation.A diet rich in antioxidants significantly reduces the incidence of several oxidative stress-related diseases. Plant phenolics are vital human dietary secondary metabolites that exhibit antioxidant properties and enhance human health. Plant phenolics are effortlessly absorbed in the intestine with relatively low toxicity and cost ,120. PheFurthermore, essential trace minerals in PEE and CWE empower their antioxidant potential. K, Ca, Fe, Mg, Cu, Mn, Zn, and Se participate in proper protein folding and stability and activate the common antioxidant enzymes, including Cu/Zn/Mn-Fe SOD and Se-GPx, as well as catalases and peroxidases that require Fe and Mg ,126,127.Moreover, the current study revealed that long-term Dexa administration might indirectly promote male infertility via hyperglycemia. In contrast, administering PEE and CWE ameliorates male infertility by improving glycemic markers. Our results provide strong support for previous studies suggesting the antidiabetic actions of Purslane ,133 and In the current study, administration of PEE and CWE reduced Dexa-induced testicular abnormalities and histological changes in rats. PEE and CWE successfully ameliorated the HPG axis dysfunction by restoring the serum testosterone, LH, and FSH levels. A remarkable improvement in the spermatogenesis markers, including sperm count, motility and morphology index, fructose levels, and mannosidase activity, also was noticed compared to the Dexa-untreated group. This ameliorative effect of PEE and CWE might be due to the presence of flavonoid constituents, minerals, and vitamins that improve the antioxidant levels and have a positive effect on the male reproductive activity. In addition, the CWE-administered groups showed improved fructose levels, androgenicity, and glycemic markers compared to PEE, which may be due to the presence of inulin. This finding was in line with the finding of Dorostghoal, Seyyednejad , where CMoreover, the effect of PEE and CWE on ER stress and autophagy has not been studied yet. The current study finds that PEE and CWE suppressed ER stress and autophagosome formation and significantly attenuated Dexa-induced testicular toxicity. The current treatments introduce a significant decline in GRP78 and Sigma 1R levels. These findings suggest that pretreatment with PEE and CWE can act as ER stress inhibitors, significantly suppress Dexa-induced autophagy, and reduce the induced testicular damage. Furthermore, the results demonstrated that the expression levels of P-mTOR(S2448) and P62 were enhanced by PEE and CWE, leading to decreased expression of ATG5, ATG7, PI3KC3, Beclin-1, and LC3II/LC3I. Previous studies are consistent with the current findings where the inhibition of ER stress can significantly inhibit autophagy’s overactivation and ultimately reduce testicular damage ,104,107.PEE and CWE are multitarget agents whose pharmacological response modulates numerous targets/pathways, which may be beneficial against male infertility. Therefore, we evaluated the protective effect of the combination between PEE and CWE against Dexa-induced testicular toxicity. CI values indicated that this combination exhibited synergistic action (CI ˂ 1) in most studied parameters. As previously published, PEE is an effective antioxidant and hypoglycemic mediator . In compDexa was found to negatively regulate the HPG axis and suppress the secretion of LH and FSH, which reduced testosterone production, consequently leading to depletion of the fructose level, and finally impairing the spermatogenesis function and the normal sperm motility. Interestingly, the current findings provide novel insight into the mechanism of Dexa-induced testicular dysfunction in male rats. Besides the androgen deficiency, oxidative stress, ER stress, and autophagy hyperactivation play a crucial role in Dexa-induced testicular toxicity. Inhibiting oxidative stress and ER stress (reduced GRP87 and Sigma 1R) using PEE and CWE remarkably blocked the Dexa-induced autophagy (elevated the P-mTOR(S2448) and P62 levels and reduced the ATG5, ATG7, PI3KC3, Beclin-1, and LC3II/LC3I expression levels). A combination of PEE/CWE proposed a synergistic (CI < 1) antioxidant and ameliorating capacity. Therefore, from a clinical perspective, combinatorial therapy of PEE/CWE along with GCs may show improved results for managing male reproductive health and delay the progression of testicular toxicity. As the mechanism of Dexa-induced autophagy is not fully elucidated, the present communication proposes enhanced oxidative stress to be an important factor, besides hormonal imbalance, in mediating Dexa-induced testicular toxicity. However, deeper insights into the autophagic and apoptotic pathways operating therein are required for a broader elucidation of the mechanism(s) by which oxidative stress augments the Dexa-induced testicular toxicity.


Myelofibrosis (MF) is the most symptomatic form of myeloproliferative neoplasm and carries the worst outcome. Allogeneic hematopoietic stem cell transplantation is the only therapy with potential for cure at present, but is limited by significant mortality and morbidity. JAK inhibition is the mainstay of treatment for intermediate- and high-risk MF. Ruxolitinib is the most widely used JAK1/2 inhibitor and provides durable effects in controlling symptom burden and spleen volumes. Nevertheless, ruxolitinib may not adequately address the underlying disease biology. Its effects on mutant allele burden, bone marrow fibrosis, and the prevention of leukemic transformation are minimal. Multiple small molecules are being tested in multiple phase 2 and 3 studies as either monotherapy or in combination with JAK2 inhibitors. In this review, the role of LSD1/KDM1A inhibition as a potential disease-modification strategy in patients with myelofibrosis is described and discussed. JAK2, CALR, and MPL. PMF is characterized by reticulin fibrosis, abnormal cytokine-mediated systemic symptoms, anemia, hepatosplenomegaly, and a propensity for progression to AML. Patients with PV and ET may progress to post-PV (PPV) and post-ET (PET)—myelofibrosis (MF), respectively. The incidence of PV, ET, and PMF is approximately 0.4 to 2.8, 0.38 to 1.7, and 0.1 to 1.0 per 100,000 persons per year, respectively [Myeloproliferative neoplasms (MPNs) are unique hematopoietic stem cell disorders sharing mutations that constitutively activate the signal-transduction pathways involved in hematopoiesis . They arectively . Median ectively ,7,8. Allectively . Thus, Jectively ,14,15,16ectively ,18,19,20ectively . In thisTET2, DNMT3A, IDH1/2), histone modification genes , RNA splicing factors , and transcription factors  have been described in MPNs [TET2 (10–20% of MPNs) and DNMT3A (5–10% of MPNs) have been found to precede JAK2V617F mutations, and have a central role in self-renewal and disease initiation in MPN hematopoietic stem cells (HSCs) [DNMT3A and TET2 in the progression to MF or secondary AML is yet to be elucidated. EZH2 mutations are seen in 5% to 10% of PMF and portend a poor prognosis [Ezh2 loss modifies phenotype and is associated with disease progression [ASXL are seen in 25% of PMF and are associated with worse outcomes [ASXL1 are associated with a higher risk of leukemic transformation. In murine models, Asxl1 loss is associated with dysplasia, cytopenia, and defective HSC self-renewal [SRSF2 mutations are mostly restricted to ET and PMF and associated with poor outcomes [TP53 mutations are uncommon in PMF or other MPNs in the chronic phase. However, they are found in up to 50% of patients with secondary AML and are often associated with ASXL1, SRSF2, IDH1/2, CBL, and LNK mutations [DNMT3A mutations. Other mutations associated with late events in the clonal progression of MPNs and secondary AML include RUNX1, FLT3-ITD, NRAS, NF1, IKZF1 and CUX1 [With the advent of next-generation sequencing and whole-genome analyses in myeloid malignancies, mutations in DNA methylation genes  ,30,31,32rognosis . In murigression ,35,36. Moutcomes ,38,39. L-renewal ,41. SRSFoutcomes . They arutations ,44. Theyand CUX1 ,46. Mutaand CUX1 ,47,48,49LSD1 is an essential gene, the loss of which leads to early embryonic lethality [LSD1 knockdown using a doxycycline-inducible short hairpin LSD1 (shLSD1) established LSD1 as a central regulator of HSPCs [LSD1 knockdown resulted in profound but reversible thrombocytopenia, neutropenia, and anemia with concurrent monocytosis. LSD1 knockdown for 27 days led to an increase in circulating multipotent progenitors and HSCs with a concomitant downregulation of chemokine (C-X-C motif) receptor 4 (CXCR4) without affecting the size of the quiescent long-term hematopoietic stem cell (HSC) pool [ethality ,51. The ethality . Conditiof HSPCs . An induSC) pool .LSD1 is a key regulator of the progression from pluripotency to terminal differentiation and balancing self-renewal and proliferation ,54. LSD1Lysine methyltransferases and demethylases are able to catalyze N-methylation and N-demthylation of histone (H) lysines (K) ,57. LSD1Histone and nucleic acid modifications provide binding sites for proteins and components of the transcriptional machinery that affect transcriptional gene silencing or activation. Histone modifications include acetylation (Ac), methylation (Me), phosphorylation (Ph), and ubiquitination (Ub). LSD1 acts as an epigenetic regulator of gene expression by altering the local state of the chromatin. Inhibition of LSD1 results in alteration of gene expression and inhibits the maturation of JAK-STAT activated megakaryocytes and myeloid cells from their progenitors. This also results in the inhibition of self-renewal potential of HSPCs harboring the pathogenic driver mutations ,63,64.LSD also regulates nonhistone substrates . LSD1 isLSD1 messenger RNA (mRNA) and excess LSD1 protein have been observed in various malignancies, including neuroblastoma, squamous-cell carcinoma, Ewing sarcoma, AML, neuroendocrine tumors, breast cancer, prostate cancer, bladder cancer, small-cell lung cancer, and colorectal cancer [JAK2V617F, CALR, and MPL activate JAK-STAT signaling via phosphorylation of STAT3, STAT5, and transcription factors, which activate specific genes with pleiotropic effects [JAK2V617F, MplW515L). LSD1 inhibition in MplW515L mice markedly suppressed myeloproliferation, reducing leukocyte and platelet counts. Spleens in animals treated with LSDi showed a dose-proportional decrease in weight. Histologically, a marked reduction in myeloid proliferation was demonstrated in the bone marrow and the spleen alongside a reversal of extramedullary hematopoiesis (EMH). Intriguingly, there was marked reduction in the degree of bone marrow reticulin fibrosis with LSD1 inhibition. LSD1 inhibition also significantly reduced serum inflammatory cytokine concentrations, in particular the plasma concentration of the chemokine (C-X-C motif) ligand 5 (Cxcl5 or IL-8 in humans), a key mediator of the inflammatory state seen in MPNs. In these mouse models, a reduction in mutant allele burden of driver mutations was also demonstrated. The observation of reduction in the degree of marrow fibrosis and the mutant allele burden of driver genes support the proposition that targeting LSD1 may induce disease modification in MPNs.Overexpression of l cancer ,72,73,74l cancer . Treatmel cancer ,77,78. Il cancer ,80. LSD1 effects . STAT3 a effects . Proof-oJAK2V617F-postive MPN mice, daily dosing improved blood counts, reduced spleen volumes, reduced marrow reticulin fibrosis, and reduced mutant allele frequencies [Given the important roles that LSD1 plays in carcinogenesis, various LSD1 inhibitors have been evaluated in clinical trials. Some of the reported LSD1 inhibitors include tranylcypromine (TCP or PCPA), ORY-1001 (iadademstat), GSK-2879552, IMG-7289 (bomedemstat), INCB059872, CC-90011, and ORY-2001 (vafidemstat). Bomedemstat  is the most extensively evaluated LSD1 inhibitor in myeloproliferative neoplasms. In quencies .Bomedemstat is the only LSD1 inhibitor clinically evaluated in patients with advanced myelofibrosis. In an ongoing phase 2 study in 89 patients with advanced MF, the efficacy and safety of bomedemstat was confirmed ,85. EighAs we progress to the era of novel therapies that could alter disease biology, there is a need to utilize end points that inform us how these novel agents could alter the disease trajectory . The cur


Cunninghamia lanceolata (C. lanceolata) belongs to Gymnospermae, which are fast-growing and have desirable wood properties. However, C. lanceolata’s stress resistance is little understood. To unravel the physiological and molecular regulation mechanisms under environmental stresses in the typical gymnosperm species of C. lanceolata, three-year-old plants were exposed to simulated drought stress (polyethylene glycol 8000), salicylic acid, and cold treatment at 4 °C for 8 h, 32 h, and 56 h, respectively. Regarding the physiological traits, we observed a decreased protein content and increased peroxidase upon salicylic acid and polyethylene glycol treatment. Superoxide dismutase activity either decreased or increased at first and then returned to normal under the stresses. Regarding the molecular regulation, we used both nanopore direct RNA sequencing and short-read sequencing to reveal a total of 5646 differentially expressed genes in response to different stresses, of which most had functions in lignin catabolism, pectin catabolism, and xylan metabolism, indicating that the development of stem-differentiating xylem was affected upon stress treatment. Finally, we identified a total of 51 AP2/ERF, 29 NAC, and 37 WRKY transcript factors in C. lanceolata. The expression of most of the NAC TFs increased under cold stress, and the expression of most of the WRKY TFs increased under cold and SA stress. These results revealed the transcriptomics responses in C. lanceolata to short-term stresses under this study’s experimental conditions and provide preliminary clues about stem-differentiating xylem changes associated with different stresses. Plants, as sessile organisms, experience constant environmental stresses throughout their life cycle . Exposurβ-aminobutyric acid and salicylic acid (SA), can induce plants to generate higher expression of stress-response genes to protect against future abiotic stress [AtLTI30, NFYA5, AtNAC72 (RD26), AtNAC19, and AtNAC55 are important drought-response genes in Arabidopsis [OsNAC14 and OsTPP3 enhance drought tolerance in Oryza sativa [PtrNAC006, PtrNAC007, and PtrNAC120 results in obvious drought tolerance in Populus trichocarpa [OsNPR1 and OsWRKY45 play complementary roles in the SA pathway in response to environmental stresses [CBF genes that regulate 414 cold-responsive (COR) genes [IbCBF3 enhances cold and drought tolerance in potato, and OsSMP1 increases cold tolerance in rice [Extreme drought and temperature conditions have long been known to have important effects on plants . Droughtc stress ,11,12,13bidopsis ,15,16,17a sativa ,19. Overchocarpa ,23,24,25tNAC72 RD, AtNAC19stresses ,28. ColdR) genes . IbCBF3  in rice .AP2/ERF, ARF, bHLH, bZIP, C2H2, Dof, HSF, MYB, NAC, and WRKY [Petunia hybrida [Arabidopsis [2-H2 or C2-HC zinc finger structural domain in the C-terminus [PtrMYB3 and PtrMYB20 of MYB TFs were found to be involved in the biosynthesis of secondary xylem [PtaERF1 has a potential role in phloem development [Transcription factors (TFs) play a pivotal role in plant signaling pathways for plant responses to biotic and abiotic stress, and also play an important role in responses to internal signals of interacting networks among developmental processes ,33,34. Land WRKY . AP2/ERFand WRKY . The fir hybrida  and the bidopsis . The WRKterminus . Accorditerminus ; additiory xylem ,40. PtaEelopment . Severalelopment .MYB2, MYB21, and MYB092 are differentially expressed upon compression stress in Cunninghamia lanceolata (Lamb.) Hook (C. lanceolata) [C. lanceolata, as endemic and evergreen coniferous species, is one of the most important artificial timber forest species in southern China because of its high timber quality and fast growth [C. lanceolata is gradually suffering from increased stress, including temperature [C. lanceolata. We hypothesized that C. lanceolata, as a gymnosperm plant, has certain tolerance mechanics in response to different stresses, which could be applied to improve the stress tolerance for plants. Therefore, we investigated three-year-old C. lanceolata trees that were exposed to simulated drought stress (20% (w/v) PEG-8000), 5 mM SA, and 4 °C cold treatment in order to reveal the potential response patterns in this gymnosperm species. In this work, we also revealed the changes in AP2/ERF, NAC, and WRKY TFs upon stress treatment. These results provide basic clues regarding how to enhance stress tolerance in C. lanceolata.Our previous study showed that ceolata) . C. lanct growth ,44. C. lperature , droughtperature , and autperature . HoweverSimulated drought stress (PEG), SA treatment, and low temperatures stimulated different changes in the soluble protein content, MDA, POD activity, and SOD activity across four time points  . The PEGC. lanceolata has a large genome size, its reference sequence and gene annotation are not available. In order to obtain a high-quality annotated sequence of the reference transcripts, we performed nanopore DRS to obtain a high-quality long transcript reference sequence. Using nanopore direct RNA sequencing, 1,031,529 reads were generated. In order to ensure the accuracy of the sequence, we used RNA-seq reads from Illumina transcriptome data to correct the long reads before UniGene clustering. Finally, 15,348 genes including 62,558 transcript isoforms were obtained. Due to the depth of RNA direct sequencing, some low-abundance genes might have been omitted. The same samples of this project were deeply sequenced using Illumina RNA-seq. Finally, we obtained a total of 22,026 genes as the reference transcripts.We collected the secondary xylem materials upon stress treatment including PEG, SA, and low-temperature treatments at 0 h, 8 h, 32 h, and 56 h . Three bp-values less than 0.05 were considered as DEGs. Under the cold treatment, we identified 1776, 1531, and 1626 DEGs at 8 h, 32 h, and 56 h, respectively  showed that three biological repeats of each treatment were clustered near each other, which reflected the reliability of the high-throughput sequencing dataset A. The coectively . Under tfamilies B, includA total of 5646 DEGs were found from all the stress treatments. Noticeably, the GO analysis showed that these DEGs were enriched in the pathway of lignin catabolism, pyrimidine metabolism, and xyloglucan metabolism C–E.TCONS_00011519. The GO enrichment analysis revealed that the genes in cluster 2 were mainly in the phosphoprotein phosphatase and abscisic acid (ABA)-mediated signal pathway  under three different stress treatments. According to the co-expression profiles of the differentially expressed genes under the different stresses and treatment points, they could be divided into 330 clusters. Among these, cluster 2 was the category with the maximum number of genes, which contained 617 DEGs, showing a rapid up-regulated expression at 8 h of cold treatment , such as pathway B. The ge pathway . The GO  pathway D. The ex pathway , which i pathway F. In con pathway , while t pathway H. In sumC. lanceolata  that was homologous with CBF genes , which was consistent with the RT-qPCR verification  family, the NAC family, and the WRKY family.In this study, we found that drought stress could stimulate significant changes in protein content, SOD, and POD. The SA and cold treatments only caused subtle changes in protein content, SOD, MDA, and POD, which then returned to normal levels. A total of 5646 DEGs were found under all the stress treatments in Larix principis-rupprechtii seedlings [Plants constantly monitor and cope with fluctuating environments, such as drought, cold, salinity stress, and so on . Droughteedlings  and cotteedlings  under drBrassica napus [C. lanceolata, the short-term cold treatment did not cause any significant changes in the soluble protein content, MDA, and POD, and only significantly increased SOD at 8 h of cold treatment  genes in Arabidopsis [C. lanceolata, most AP2/ERF TFs belong to the ERF subfamily and the DREB subfamily  control (untreated plants), (2) simulated drought stress treatment with 20% (w/v) PEG-8000, (3) exogenous 5 mM salicylic acid (SA), and (4) cold treatment in a growth chamber at 4 °C. In order to avoid the effect of circadian rhythms on gene expression, we collected materials at 24 h intervals. Thus, all the plants under the different stresses were treated at ~8:00 a.m., and we harvested secondary xylem and leaves after 0 h, 8 h, 32 h, and 56 h. In total, we selected nine individual plants as one biological repeat for one time interval for each treatment. In total, three biological repeats were collected. The above materials were immediately frozen in liquid nitrogen and stored at −80 °C until use for subsequent high-throughput sequencing and real-time quantitative PCR (RT-qPCR). Three biological repeats were used for sequencing and qPCR.The frozen leaves (0.1g) with three biological repeats were crushed into a fine powder using a tissue crusher , and then suspended in 0.9 mL PBS solution  in a volume ratio of 1:9. The homogenate was centrifuged at 4 °C with 4000 rpm for 10 min. The clear supernatant was used for the determination of MDA, protein content, SOD, and POD using commercial assay kits . The protein contents were detected using a Thermo Fisher Multiskan™ FC microplate photometer and determined using the total protein assay kit (with the standard BCA method) ; the protein concentration was calculated by measuring the absorbance in 562 nm. We took the protein content changes as physiological traits since the protein content was used in the determination of SOD and POD activity as described in the total superoxide dismutase (T-SOD) assay kit (hydroxylamine method)  and the peroxidase assay kit . The activity of MDA was determined using an MDA assay kit  . The actTotal RNAs were extracted from secondary xylem materials treated with SA, PEG, and cold stress at four time points  using the RNAprep Pure Plant Kit . RNA quality was determined using a NanoDrop 2000c UV–Vis spectrophotometer  followed by 1% agarose gel electrophoresis. High-quality total RNAs with RNA integrity number (RIN) values higher than 8 were used in subsequent Illumina RNA-seq, and RT-qPCR. In this study, we constructed strand-specific RNA-Seq libraries including the control and the PEG, SA, and low-temperature treatments for 0 h, 8 h, 32 h, and 56 h. The RNA-Seq was constructed using the deoxyuridine triphosphate (dUTP) method using Illumina Adapter Sequences (5’ AATGATACGGCGACCACCGA GATCTACACIIIIIIIIAC ACTCTTTCCCTACACGACGC TCTTCCGATCT (-) AGATCGGAAGAGCACACGTC TGAACTCCAGTCACIIIIII IIATCTCGTATGCCGTCTTCTG CTTG 3’) and the Illumina TruSeq Stranded mRNA kits. Three biological repeats were sequenced for each treatment based on the Illumina HiSeq platform  using the 150 bp paired-end strategy. https://github.com/PrincetonUniversity/DP_GP_cluster (accessed on 9 September 2019)) performs very well in clustering gene expression time-series data using an infinite Gaussian process mixture model. After we obtained the DEGs, we extracted the FPKM values of the DEGs. Then, we used the FPKM values as inputs for the DPGP to cluster with the default parameters so as to identify similar expression disjoint clusters.The low-quality reads were removed using the ht2-filter from HTQC . Then, wC. lanceolata was not available due to its large size. Thus, we also used long-read sequencing based on nanopore direct RNA-seq (DRS) to generate a high quantity of unigenes and to improve the integrity of the unigenes. The RNA quality was determined using a NanoDrop 2000c UV–Vis spectrophotometer  followed by 1% agarose gel electrophoresis, and the high-quality total RNA with RIN values higher than 8 was used. The total RNAs of the secondary xylem from different stages and different treatments were mixed into one sample for DRS, and mRNA was isolated using the Dynabeads mRNA Purification Kit . The DRS library was built using the SQK-RNA002 Direct RNA Sequencing Kit (Oxford Nanopore Technologies). The constructed library was loaded onto the R.9.4.1 FlowCell (FLO-MIN106) for sequencing in a MinION MK 1B sequencer running for 48 h. After sequencing, the electronic raw signals were identified using Guppy (version 2.3.1) with the default parameters. The transcriptome data generated by DRS were corrected by lordec-correct (-k 21 -s 3) [The genome of 21 -s 3)  using RN21 -s 3)  to removIn this study, the gene ontology (GO) terms of each unigene were obtained using BLAST2GO . The GO A total of 960 TFs were identified using the iTAK  database® Green premix ProTaq HS qPCR Kit (Accurate Biotechnology (Human) Co., Ltd., Changsha, China). The instrument was the Applied Biosystems QuantStudio™ 6 Flex Real-Time PCR System . Ribosomal 5S (TCONS_00059130) was used as a positive control for the validation of each gene. Three biological repeats were measured for each stress condition.RT-qPCR was used to verify the gene expression patterns of the AP2 family, the NAC family, and the WRKY family under drought, low-temperature, and SA treatments. Premier 5 was used to design the primers . All theC. lanceolata upon drought, SA, and cold treatment at different time points (C. lanceolata. These results revealed the physiological and transcriptomics responses to short-term stresses in C. lanceolata and provide preliminary clues about SDX changes associated with different types of stress. Additionally, our work also provides a molecular direction for future studies regarding the screening of resistance genes in typical gymnosperm species.In this study, we investigated the changes in physiological markers in e points . Moreove


Epithelial–mesenchymal transition (EMT) plays a crucial role in appropriate embryonic development, as well as wound healing, organ fibrosis, and cancer progression. During cancer progression, EMT is associated with the invasion, metastasis, and generation of circulating tumor cells and cancer stem cells, as well as resistance to chemo- and radiation therapy. EMT is induced by several transcription factors, known as EMT transcription factors (EMT-TFs). In nearly all cases, EMT-TFs appear to be regulated by growth factors or cytokines and extracellular matrix components. Among these factors, transforming growth factor (TGF)-β acts as the key mediator for EMT during physiological and pathological processes. TGF-β can initiate and maintain EMT by activating intracellular/intercellular signaling pathways and transcriptional factors. Recent studies have provided new insights into the molecular mechanisms underlying sustained EMT in aggressive cancer cells, EMT induced by TGF-β, and crosstalk between TGF-β and growth factors. Smad4 gene are observed in approximately 60% and 30% of pancreatic and colorectal cancers, respectively. Genetic mutations and repression of TGF-β receptors and other Smads are also occasionally observed in various cancers. Thus, TGF-β is considered to act as a cancer suppressor in the early stages of cancer (Transforming growth factor-β (TGF-β), a prototypical member of the TGF-β family, binds to transmembrane serine-threonine kinase receptors type Ⅰ (TβR-Ⅰ) and type Ⅱ (TβR-Ⅱ), and transduces canonical signals through Smad proteins ,2. TGF-βf cancer .In the advanced stages of cancer, cancer tissues contain a high amount of TGF-β . TGF-β aEMT was initially described by developmental biologists, and now is known as a phenotypic conversion that facilitates embryonic development . During In adults, the EMT program plays crucial roles in pathological and physiological processes, including wound healing, tissue fibrosis, and cancer progression. Keratinocytes adjacent to the wound site start to migrate toward the wound center after they undergo morphological and adhesive changes which resemble those that occur during EMT. In chronic progressive fibrosis of the kidneys and liver in mice, approximately 35% of fibroblasts originated from the epithelial and endothelial cells through EMT, as determined by using a sophisticated mouse model . EMT in EMT has also been studied extensively in cancer and is believed to promote chemo-resistance, invasion, and metastasis, and generate circulating tumor cells (CTCs), cancer stem cells (CSCs), and aggressive cancer cells. Surviving CTC is known to express both epithelial and mesenchymal markers, such as E-cadherin and N-cadherin, respectively, and is termed Type III EMT or partial EMT . When CTInterestingly, a heterophilic cell–cell adhesion between E-cadherin on the cancer cell membrane and N-cadherin on the membrane of fibroblasts or cancer-associated fibroblasts (CAFs) enables fibroblast-led collective cancer cell migration ,14. MoreK-RAS mutations are very frequently detected. During the early stages of carcinogenesis, active RAS signals result in the sustained activation of MAPK pathways, which reportedly help overcome the cancer suppressing effects of TGF-β by promoting Smad degradation  and efficient invasion into stromal tissues, possibly leading to single cancer cell migration.Collective cancer cell migration exhibits multicellular clusters resembling a bunch of grapes and depends on cell–cell adhesion through epithelial adhesion molecules of solid cancer cells . On the radation  16]. Wh. WhK-RASto TGF-β . Thus, tIt is widely known that both “complete EMT” and “partial EMT” are induced by several common transcription factors, referred to as EMT-TFs. Representative EMT-TFs include the basic helix–loop–helix (bHLH) factor TWIST (TWIST1 and TWIST2), the SNAIL family of zinc finger transcription factors , and the two-handed zinc finger factors of the zinc finger E–box-binding homeobox (ZEB) family proteins  .Drosophila as a zygotic gene required for dorsoventral patterning, and its gene mutation is reported to cause Saethre Chotzen Syndrome, an autosomal dominant human craniosynostosis syndrome [Twist1 null mice die at approximately E11 due to severe phenotypic abnormalities including neural tube closure defects. Twist2 null mice die immediately after birth due to severe inflammation and cachexia. TWIST consists of a nuclear localization signal and two glycine-rich domains in the N-terminal region, a bHLH domain in the central region, and a WR (tryptophan and arginine) motif, also known as a Twist box, in the C-terminal region [TWIST was originally identified in syndrome . Twist1 l region ,18. The l region , while tl region . Phosphol region . Althougl region ,23. Phosl region ,25,26,27Snail gene knockout mice exhibit severe gastrulation defects, resulting in embryonic lethality at an early stage of development. The SNAIL family members consist of a SNAG domain in the N-terminal region, a nuclear export sequence and a serine-rich domain (SRD) in the central region, and several zinc finger clusters in the C-terminal region [SNAIL is widely known to be the most important transcription factor for EMT , becausel region ,30,31. Sl region ,32. SNAIl region ,34. Thesl region ,36. Furtl region .Smuc null mice show almost normal phenotypes [Similar to SNAIL, SLUG is also phosphorylated at multiple sites: Ser87 by ERK, Ser92/Ser96/Ser100/Ser104 by GSK3β, and Ser158/Ser254 by p21PAK4 ,38,39. Tenotypes . Thus, SZeb1 and Zeb2 show postnatal death with severe defects in hematopoiesis and skeletal bones, and embryonic lethality with severe defects of cranial neural crest cells, respectively. They consist of several zinc finger clusters at both the N- and C-terminal regions. Each of these two clusters located in the N- and C-terminal regions binds to the paced E-boxes in the E-cadherin promoter, and then represses the expression of E-cadherin. ZEB1/2 also directly bind to the E-box in many gene promoters through the zinc finger clusters. In the central region, ZEB1/2 have a Smad-binding domain (SBD), C-terminal binding protein (CtBP) domain, and homeodomain. The homeodomain of ZEB1/2 regulates transcriptional control by modifying the protein–protein interaction. The SBDs in ZEB1 and ZEB2 are approximately 40% similar in amino acid sequence, but have dramatically low structural similarity [The ZEB family members, ZEB1 and ZEB2 (ZEB1/2), are also known to be crucial factors for EMT, because knockout phenotypes of milarity . In respmilarity . Consequmilarity ,44. In Zmilarity .Epidermal growth factor (EGF), tumor necrosis factor-α (TNF-α), and Wnt signals are reported to cooperate with TGF-β signaling during cancer progression . The synThe underlying mechanism of enhancement of EMT by FGF2 and FGF4 is very unique. FGF receptor genes encode four functional receptors (FGFR1-FGFR4) that consist of an intracellular tyrosine kinase domain, a transmembrane domain, and three extracellular immunoglobulin-like domains  . AlternaTranscriptional repression of E-cadherin is found in various solid cancers and is sometimes mediated by EMT-TFs . Among tThe pathological roles of SNAIL and ZEB1/2 are still controversial during cancer progression. In a mouse pancreas cancer model, cancer aggravation, including tumor differentiation, invasion, and metastasis, is not affected by genetic SNAIL depletion, but is reduced by genetic depletion of ZEB1 ,62. HoweZEB1 promoter and induces endogenous ZEB1/2 expression in cancer cells [SNAI1-promoter and induces endogenous SNAIL expression [MEK–ERK inhibition downregulated SNAIL as well as ZEB1/2. The E26 transformation-specific (Ets) family of transcription factors has twenty-eight members in the human genome, and regulates many different biological and pathological processes ,65. Ets er cells . In addipression . Thus, Epression . ESE1 ispression ,67,68, wAggressive cancer cells exhibit sustained high levels of ZEB1/2. ZEB1/2 directly bind to the promoter region of ESRP1/2 and transcriptionally suppress them, leading to regulating alternative splicing machinery of various mRNAs, including FGFRs, CD44, Mena (a member of the Enabled (Ena)/vasodilator-stimulated phosphoprotein family of proteins), and Rac1. As described above, when ZEB1/2 were upregulated and maintained at high levels in aggressive cancer cells, the IIIc isoforms of FGFRs were expressed through isoform switching when ESRP1/2 expression was repressed. Due to preferential binding of FGF2 and FGF4 to the IIIc-isoforms of FGFRs, the cancer cells expressing IIIc isoforms of FGFRs become sensitive to FGF2 and FGF4, which are abundant in stromal tissues. FGF2/4-bound FGFRs activate ERK pathways and maintain high levels of ZEB1/2 expression via activating ETS pathways, leading to a positive feedback loop of ERK–ZEB1/2–ESRP1/2–FGFRIIIc. Indeed, a positive correlation between ZEB1/2 and IIIc isoforms of FGFRs is observed in breast cancer cells, especially the basal-like subtype of breast cancer cells, such as BT549, MDA-MB-231, and Hs578T cells. These cells, with high ZEB1/2 levels, expressed only IIIc isoforms of FGFRs, with low ESRP1/2 levels  Figure .Hyaluronic acid, also called hyaluronan, is a glycosaminoglycan distributed widely throughout epithelial and stromal tissues. Hyaluronan is a major component of extracellular matrices, and has pivotal roles in cell proliferation, migration, and invasion during cancer progression, through its binding to cell surface receptors, CD44. It is reported that CD44 has an affinity towards other ligands such as fibronectin, collagen, chondroitin, and sulfated proteoglycan . IsoformSince EMT processes occur in normal embryogenesis, normal fibroblasts and some stromal cells constitutively express EMT-TFs at high levels. This makes it hard to use EMT-TFs as diagnostic markers that specifically detect cancer cells undergoing EMT or partial EMT. During EMT, a large number of mRNAs are changed by alternative splicing, producing divergent gene products . Thus, tIt has become increasingly clear that the expression of EMT-TFs is regulated transcriptionally by secreted factors and extracellular matrices, post-transcriptionally by several microRNAs, and post-translationally by ubiquitin-dependent degradation. Additionally, the function of EMT-TFs is controlled by various post-translational modifications. Consequently, EMT-TFs modify the expression of EMT marker proteins at the transcriptional and epigenetic levels. TGF-β autonomously secreted from cancer cells regulates the expression of EMT-TFs and acts as a cancer promoter in cooperation with active K-RAS mutants and other factors secreted from stromal cells in the cancer microenvironment. Thus far, it is still unclear to what extent cells undergo a conversion of cell morphology by partial EMT in vivo during cancer progression. In addition, recent studies have demonstrated that a partial EMT-like morphology or expression of EMT-TFs is associated with CTC and CSC. Therefore, understanding the molecules underlying partial EMT would be clinically important for both the diagnosis and treatment of cancer.


In multivariate analysis adjusted for subtype and grade, gp78 protein is an independent predictor of poor outcomes in women of African ancestry. Furthermore, gene expression signatures, derived from patients stratified by gp78 protein expression, are strong predictors of recurrence and pathological complete response in retrospective clinical trial data and share many common features with gene sets previously identified to be overrepresented in breast cancers based on race. These findings implicate a prominent role for gp78 in tumor progression and offer insights into our understanding of racial differences in breast cancer outcomes.Women of African ancestry suffer higher rates of breast cancer mortality compared with all other groups in the United States. Though the precise reasons for these disparities remain unclear, many recent studies have implicated a role for differences in tumor biology. Using an epitope-validated antibody against the endoplasmic reticulum–associated E3 ligase, gp78, we show that elevated levels of gp78 in patient breast cancer cells predict poor survival. Moreover, high levels of gp78 are associated with poor outcomes in both ER  In 2021, breast cancer became the most frequently diagnosed malignancy worldwide –5. This The protein gp78 is an endoplasmic reticulum (ER) resident E3 ligase that targets numerous proteins for degradation by ubiquitylation and, through its cytoplasmic E3 ligase domain, plays a major role in the unfolded protein response (UPR) and other adaptive cellular responses to ER stress –15. BecaIn addition to its role as an ER-associated E3 ligase, gp78 has also been linked, in past studies, to binding activity for the autocrine motility factor (AMF) . This asUsing an epitope-defined antibody raised against a known peptide sequence in the C-terminus of gp78 , 14, 24,In this current study, we assess the role of gp78 in breast cancer by characterizing its protein expression in a large (>500 patients) cohort of patients with breast cancer and defining its association with patient and tumor characteristics and features, including survival, grade, subtype, race/ethnicity (self-identified), and gene expression. The analysis confirms that gp78 is expressed at a higher level in the breast cancers of women of African descent, is associated with gene expression patterns that are predictive of tumor recurrence and response to therapy, and demonstrates that gp78 independently predicts poor breast cancer survival in women of African ancestry even after adjusting for age, stage, grade, and subtype.n = 560) residing in a designated health disparities catchment area in Eastern North Carolina  and arrayed in tissue microarray (TMA) format  (R2 = 0.93) (https://doi.org/10.1172/jci.insight.157465DS1). Compared with normal breast, gp78 protein is expressed at substantially higher levels in breast cancer  format , 49, 50 +, or 3+ , 49, 51.1 [%1+]) , 49. Usit cancer . When cot cancer , left. Nt cancer , right. t cancer  . Analysis by gene set enrichment profiling, based on the differential gene expression pattern of patients stratified by gp78 H-score using the optimal cutoff of all samples, shows enrichment for multiple gene sets representing pathways important in cellular immune responses, stress responses, metabolism, cell cycle, and cellular signaling (RNF45) demonstrates a comparable overlap with the TCGA breast cancer GSEA stratified by race (race-based TCGA breast cancer GSEA and the gp78 protein-based GSEA (Pearson’s r = 0.5567), is higher compared with the gp78 mRNA-based GSEA (Pearson’s r = –0.1195) . Sp. Spn = 1ignaling . Interesignaling . Gene seignaling , and thoignaling , demonstignaling . In contignaling . Interes by race . However–0.1195) . This is–0.1195) . Finally–0.1195) .To define both patient and tumor parameters that are associated with increased expression of gp78, we profiled tumor and patient characteristics comparing biomarkers commonly associated with more aggressive forms of breast cancer by unsupervised hierarchical clustering . This anUsing the concept of coexpression modules or lists of genes with highly correlated expression –60, we lHierarchical clustering analysis, comparing patient gp78 gene module scores with other previously described gene signatures –60, demo+ CD8 T cells and CD68 macrophages in close spatial proximity with tumor, although there was nonsignificant proximity of CD8 and CD68 cells with PD-L1+ tumor . Furthermore, greater than 70% of the gene sets, enriched by gp78 protein expression, are also enriched in patients with breast cancer, stratified by race, described in a previous study using TCGA data , this conclusion is based on an antibody (3F3A) that recognizes an unknown epitope . Direct hi tumors may have arisen in early ancestral African populations as a feature with universally associated aggressive breast cancers regardless of subtype prior to the evolution of TNBC, where gp78hi tumors may have been a more predominant subtype prior to the out-of-African events believed to occur in the late Pleistocene epoch  tissue samples and deidentified clinical information abstracted from the medical records were requisitioned and initially procured for patients with breast cancer who underwent surgery for Stage 0 to Stage IV breast cancer between 2001 and 2010 at Pitt County Memorial Hospital  in Greenville, North Carolina, USA. Race, ethnicity, or ancestry was self-reported at the initial visit and captured in the medical record. Survival was recorded retrospectively from the medical records and the cancer registry. The median follow-up was 8.5 years. Tissue microarrays were constructed using 1 mm cores as previously described , 95, witBreast tumor tissue microarrays were stained, using monoclonal primary antibodies, ERα at 1:35, low pH ; EGFR at 1:500, high pH ; E-Cadherin at 1:50, high pH ; Ki-67 ready to use, at low pH ; and GATA3 at 1:50, high pH . Also, polyclonal primary antibodies were used, FOXA1 at 1:10,000, high pH  and the ready-to-use antibody c-erbB–oncoprotein at a high pH . The gp78 antibodies were generated and affinity-purified from rabbits injected with a peptide containing aa 574–597 derived from the C-terminus of gp78, used at a final concentration of 0.4 μg/mL, high pH; LC3A/B antibodies (cross-reacting with both LC3 A and B isoforms) used at a final dilution of 1:3000, high pH ; antibodies to androgen receptor  used at 1:1000, high pH; and Kaiso antibodies used at 1:1000 dilution, high pH, as previously described .+ when available information in the patient medical record showed a score greater than 2 by in situ hybridization or 3+ by IHC. ER+ patients were classified from the medical record and confirmed by IHC; missing data were replaced by TMAMedian follow-up and median survival for patients were 8.5 and 6.67 years, respectively. Clinical subtypes 2 or 3 were categorized as HER2All digital scoring was performed using the nuclear and membrane algorithms provided by the Leica Biosystems Aperio software. Areas of interest were outlined by the pathologist and then scored independently by the pathologist and the respective algorithms.We use the Ultivue UltiMapper I/O PD-L1 assay and the PD-1 assay to collect the qmIF data. This PD-L1 kit uses the following antigens: CD8, CD68, PD-L1, pan-cytokeratin (panCK), and DAPI (DNA marker). The PD-1 immune exhaustion kit uses a 4-plex panel for CD3, CD45RO, PD-1, panCK, and DAPI. The raw image data were collected at 20×. The fluorescent dye intensities are normalized to 0–255. Image analysis was performed using a commercial software package  at full magnification. Point cloud generation and nearest-neighbor analysis were conducted as previously described .2 test. Comparison of IHC scoring was performed by a 2-tailed t test and plotted as previously described (n = 1000) using the pvclust R package  value for each cluster in a dendrogram . The P value–ranked gene list was used for functional correlation using preranked GSEA software (http://software.broadinstitute.org/gsea/index.jsp). Genetic admixture analysis was conduct as previously described . A 2-tailed t test was employed to test the null hypothesis (H0) assumption of equality of the protein values in 2 defined groups of data and is demonstrated by violin plots using R software and ggplot2 package (A Spearman’s rank correlation test was performed to test the relation between its protein H-score and gene expression (RPKM) values . A compl package .https://cran.r-project.org/web/packages/survival/survival.pdf). The significance of individual hazard ratios was estimated by Wald’s test.To classify the patients into low- versus high-risk categories using selected protein H-scores, the optimal cutoff approach  has beenUse of human tissue in this study was reviewed and approved by the IRBs of the Intramural Research Program of the NIH, East Carolina University, and Columbia University Irving Medical Center.SKS, YCT, AMW, NV, and KG formulated and designed the study; SKS, JM, TY, and SB reviewed performed biostatistical analysis; SKS, KG, RY, and JSB wrote the manuscript; JSB, AC, SGH, JP, and JW performed staining and scoring; EJPS, AMN, ADS, CY, MBD, and VM provided scientific input; JM review statistical analysis; SMH provided reagents and tissue blocks; and MY generated and validated antibodies.


Neurodegenerative diseases (NDDs) are irreversible, progressive diseases with no effective treatment. The hallmark of NDDs is the aggregation of misfolded, modified proteins, which impair neuronal vulnerability and cause brain damage. The loss of synaptic connection and the progressive loss of neurons result in cognitive defects. Several dysregulated proteins and overlapping molecular mechanisms contribute to the pathophysiology of NDDs. Post-translational modifications (PTMs) are essential regulators of protein function, trafficking, and maintaining neuronal hemostasis. The conjugation of a small ubiquitin-like modifier (SUMO) is a reversible, dynamic PTM required for synaptic and cognitive function. The onset and progression of neurodegenerative diseases are associated with aberrant SUMOylation. In this review, we have summarized the role of SUMOylation in regulating critical proteins involved in the onset and progression of several NDDs. Neurodegenerative diseases (NDDs) impose a substantial medical and financial burden on society worldwide. NDDs are marked by neuronal dysfunction and death, causing progressive degeneration of the structure and function of the central and peripheral nervous system ,2,3. A lThe progressive loss of a large number of neurons and the miscommunication of neuronal circuits results in memory impairment, learning disabilities, deteriorated synaptic function and cognitive defects ,4,5,6,7.SUMOylation involves the attachment of small ubiquitin-like modifier (SUMO) proteins to the lysine residues in the protein. Meluh and Koshland first identified it in 1995 ,21,22. ISaccharomyces cerevisiae, multiple proteins involved in DNA repair pathways undergo SUMOylation in response to DNA damage signals. It expedites the DNA repair process [SUMOylation is a dynamic process involving target protein switching between SUMOylated or deSUMOylated states. SUMOylation regulates target protein trafficking and subcellular localization, stability, solubility, activity structural conformations, and protein interactions . In an u process . Similar process . AlthougSUMOylation is a reversible post-translational protein modification. Similar to ubiquitination, SUMO moieties form isopeptide bonds with the ε-amino group of acceptor target lysine residue ,31,37. SIn addition to UBC9-mediated target recognition potency, the binding affinity of UBC9 to the target sequence enhances substrate SUMOylation. Additional mechanisms further vary the pool of SUMOylated proteins in a given tissue type. These involve the external or internal stimuli-induced regulation of UBC9 expression. UBC9 activity is modulated via intrinsic post-translation modifications, such as self-SUMOylation at Lys 14 and Cdk1/Cyclin B-mediated phosphorylation . Identifψ X ψ ψ or ψ ψ X ψ. In the consensus sequence, “ψ” is the hydrophobic amino acid with a large non-polar side chain, typically Isoleucine (I), Valine (V) or Leucine (L), and X is any amino acid. The hydrophobic core is flanked on one side or the other by negative charged Aspartic acid (Asp), Glutamic acid (Glu) or Phosphoserine (Ser) residues. Hydrophobic sequences facilitate SUMO interaction. RanBP2, CBX4, SLX4 and ZNF451 are some of the more well-studied E3 ligases in this family [Based on molecular structure, the SUMO–E3 ligases can be classified mainly into the SP-RING domain family, the TRIM superfamily, SIM-containing SUMO E3 ligases and others ,42,43,44s family ,47,48,49Proteomic analysis revealed that most acceptor lysine residue is in the consensus motif. The classical consensus SUMOylation motif is ψKXE/D, where ψ represents hydrophobic residue, K is the acceptor lysine residue, X represents any amino acid, and E/D is glutamic or aspartic acid residues, respectively . ψ contrSeveral studies have established the role of SUMOylation in the onset and progression of NDDs. Aberrant SUMOylation causes the aggregation of the toxic proteins, defects in protein trafficking, the modification of the ion channel properties and the release of neurotransmitters in neurons, oxidative stress and alteration in gene expression. Altered SUMOylation potentially modifies the functions of hundreds of proteins in many different pathways, and results in cognitive deficits associated with NDDs.AD is the dominant form of escalating dementia. AD primarily occurs in an ageing population, but hereditary or non-hereditary mutations, lifestyle and environment may contribute to the onset and progression of AD pathogenicity. Currently, there are more than 50 million AD patients, and by 2050 these numbers are predicted to reach 152 million . Clinica (SNAP-23) phosphorylation, facilitating the release of UBC9 from EVs, promoting the development of AD [1-42 peptide downregulates the levels of SUMO-activating enzyme SAE2 and the SUMO E3 ligase PIAS1/2. This results in decreased levels of SUMO1 or SUMO2/3-conjugated proteins [Cell culture-based in vitro systems and a transgenic mouse model of AD were used to study the impact of SUMOylation on AD pathology. Expression studies in the early-aged Tg2576 mouse model of AD revealed no changes in the global levels of SUMO-1 . In contnt of AD . In addint of AD . Proximint of AD ,90. In cnt of AD . This rent of AD . These pproteins . Moreoveproteins . The actproteins . Furtherproteins . Pharmacproteins . This isproteins . The rolPD is a multifactorial neurodegenerative disease. It is manifested by the progressive loss of dopaminergic neurons projecting from the substantia nigra compacta to the striatum. PD-associated neurological disorders include motor and nonmotor symptoms. The early pathological symptoms are rapid eye movement, sleep behavioral disorder and decreased smell. The progression of the disease is associated with the onset of cognitive impairment and hallucinations. The hallmark of PD progression is motor dysfunction. It includes tremors, rigidity, bradykinesia, gait stiffness, hypokinesia and imbalance ,97,98. PPARK7 gene) transcriptional factors, an impaired ubiquitin-proteasome system, aberrant splicing events, mitochondrial dysfunction and neuronal oxidative stress. These could potentially lead to the deposition of Lewy bodies in neurons. Lewy bodies are the eosinophilic cytoplasmic inclusions of 5-30 µm spherical masses with a dense core surrounded by radiating fibrils. Lewy bodies are strongly stained for α-synuclein protein [SCNA gene encodes the α-synuclein protein. Autosomal dominant missense mutations in the SNCA gene lead to the early onset of PD [SCNA gene multiplication (duplication and triplication of SCNA gene) increases the dose of wildtype proteins and can cause autosomal dominant parkinsonism. A significant number of genetic, transgenic and viral-based studies in rodents and primates, using either wildtype or mutant α-synuclein protein, clearly demonstrate an α-synuclein association with the progressive degeneration of neurons, and thus its crucial role in the pathogenesis of PD. α-Synuclein oligomerization promotes its binding to the mitochondrial outer membrane translocase (TOM20) and the voltage-dependent anion channel (VDAC). It results in the inhibition of the mitochondrial import system [N-ethylmaleimide-sensitive factor attachment protein receptor) mediates membrane fusion at synaptic terminals. SNARE proteins are present on vesicles (v-SNARE) or target membranes (t-SNARE). The C-terminus of α-synuclein binds to VAMP2 (vesicle-associated membrane protein 2) and promotes SNARE complex formation. α-synuclein overexpression or aggregates inhibit v-SNARE binding to t-SNARE, which impairs neurotransmitter release and thus leads to synaptic dysfunction [PD is pathological and primarily marked by the cytoplasmic deposition of Lewy bodies, a neuronal inclusion consisting of α-synuclein protein aggregations. The intracellular aggregation of α-synuclein causes decreased membrane potential. Furthermore, it results in oxidative stress, excitotoxicity, neuroinflammation and finally, apoptosis of the neuron . The seq protein . In addi protein . SCNA geet of PD . In addit system . This evt system . α-Synuct system . Compromt system ,108. NDDfunction ,110. α-sfunction ,112. Inhfunction ,114. Drosophila model exerts neuroprotective effects [PARK2 gene) [PARK2 are the cause of autosomal recessive juvenile parkinsonism [PARK7 gene) protein contributes to detecting and modulating neuronal oxidative stress. It functions as a regulator of mitochondrial biogenesis and a stress regulator. Mutations in DJ-1 are identified in PD patients [α-Synuclein post-translational modification (PTM) may influence its toxicity and aggregation. Phosphorylation, nitration, acetylation, ubiquitination, O-GlcNAcylation and SUMOylation extensively modify α-synuclein ,117,118. effects . Lewy boK2 gene) . Mutatioinsonism . Parkin insonism . Parkin insonism . Neuronsinsonism . In addipatients ,128. Thepatients . The SUMpatients ,130. It could be hypothesized that the SUMOylation of multiple key proteins involved in mitochondrial biogenesis reinforces cell survival, whereas deSUMOylation causes PD pathogenesis. HTT) gene [HTT gene encodes the 348 kDa Huntingtin (HTT) protein found in the cytoplasm and nucleus of neurons. The HTT protein plays a critical role in microtubule-based axon trafficking, which involves nuclear–cytoplasmic protein trafficking and antero- and retrograde axonal transport [HTT mice are embryonically lethal [HTT knockout mice showed impaired vesicular and mitochondrial trafficking in both directions, and progressive brain degeneration [HTT gene with the abnormally high numbers of CAG triplet repeats (polyQ) is termed mutant HTT (mHTT). mHTT mitigates the HD disease pathology mainly in striatal medium-sized spiny neurons, cortical pyramidal neurons and thalamic neurons. However, mHTT is susceptible to aggregation, which causes neuronal dysfunction and death [Huntington’s disease is a rare, incurable, neurodegenerative, monogenetic, dominantly inherited disease. It is a polyglutamine (poly Q) disorder of Huntingtin protein (HTT). It is caused by the expansion of a CAG repeat (encoding glutamine) at the exon 1 of the Huntingtin (TT) gene ,132,133.TT) gene ,135,136.ransport ,138. Theransport . In addiransport . Homozygy lethal ,142. Conneration . The HTTnd death ,145. HD nd death ,148,149.HTT gene (HTTex1) has a paradoxical effect. It leads to a reduced ability to form aggregates and increased solubility, and promotes its ability to repress transcription [The motor symptoms include involuntary movements, impaired voluntary movements, speech difficulties and unusual eye movements, whereas cognitive symptoms include learning deficit and lack of impulse control. The neuropsychiatric symptoms include depression, insomnia, frequent fatigue and suicidal tendencies. Several post-translational modifications modify Huntingtin’s protein. Ubiquitination and SUMOylation antagonistically modify the same lysine residue ,152,153.cription . In addicription . SUMO E3cription ,157. PIAcription . Rhes , Transient receptor potential V1 (TRPV1), two-pore potassium channel (K2P1), and multiple voltage-gated potassium channels , as SUMOylation targets ,190,191.2O2) [2O2 attack the iron–sulfur centers of enzymes involved in glucose metabolism, such as complex I–III in mitochondria, and consequently inhibit their function [2O2, which transiently inactivates SUMO E1 and E2 enzymes. It induces the formation of a disulfide bond between their catalytic cysteines [Prolonged chronic hyperglycemia is associated with excessive cellular oxidative stress and impaired mitochondrial function . In vitr2O2) . ROS/H2Ofunction . This lefunction . Prolongysteines , which m


RUNX1 are associated with qualitative and/or quantitative platelet defects, and predispose to hematologic malignancies. The latter manifests in approximately 44% of carriers and can occur from early childhood to late adulthood. In addition to the predisposing RUNX1 germline variant, the acquisition of somatic genetic alterations is presumed to drive leukemic transformation in an inflammatory bone marrow niche. The spectrum of somatic mutations occurs heterogeneously between individuals, even within families, and there is no clear genotype–phenotype correlation. In this review, we summarize previously published patients harboring (likely) pathogenic RUNX1 germline alterations in whom somatic alterations were additionally analyzed. We provide an overview of their phenotypes and the most frequent somatic genetic alterations.Pathogenic germline variants affecting RUNX1 germline variants cause autosomal dominantly-inherited familial platelet disorder with predisposition to hematologic malignancies (RUNX1-FPD). RUNX1-FPD is characterized by incomplete penetrance and a broad spectrum of clinical phenotypes, even within affected families. Heterozygous RUNX1 germline variants set the basis for leukemogenesis, but, on their own, they are not transformation-sufficient. Somatically acquired secondary events targeting RUNX1 and/or other hematologic malignancy-associated genes finally lead to MDS, AML, and rarely other hematologic malignancies including lymphoid diseases. The acquisition of different somatic variants is a possible explanation for the variable penetrance and clinical heterogeneity seen in RUNX1-FPD. However, individual effects of secondary variants are not yet fully understood. Here, we review 91 cases of RUNX1-FPD patients who predominantly harbor somatic variants in genes such as RUNX1, TET2, ASXL1, BCOR, PHF6, SRSF2, NRAS, and DNMT3A. These cases illustrate the importance of secondary events in the development and progression of RUNX1-FPD-associated hematologic malignancies. The leukemia-driving interplay of predisposing germline variants and acquired variants remain to be elucidated to better understand clonal evolution and malignant transformation and finally allow risk-adapted surveillance and targeted therapeutic measures to prevent leukemia.Pathogenic loss-of-function  Three major protein isoforms of RUNX1 are known . Their expression is regulated by two different promoters . NRUNX1 germline variants, who were analyzed for additional somatic gene variants regardless of whether a somatic variant was identified or not. Somatic alterations were investigated by karyotyping, array-CGH, and/or DNA sequencing . Overall, 31 of 56 (55%) cases with given karyotype information showed a somatically abnormal karyotype, all of which were associated with HM. Interestingly, of 19 patients with acquired somatic RUNX1 mutations, 15 had an abnormal karyotype (79%). In four of these cases chromosome 21 was affected, leading to duplication of the mutated RUNX1 allele, detected via karyotyping and variant allele fraction of the RUNX1 germline variant. In contrast, only 14 (41%) cases were reported with an abnormal karyotype in 34 cases without somatic RUNX1 alterations and available karyotype information. Noteworthy, RUNX1 somatic status was not analyzed in two cases with abnormal karyotype. A summary of frequently investigated and mutated genes is given in n = 91). Overall, RUNX1 represents the most frequently analyzed gene . Other commonly analyzed genes were GATA2, PTPN11, CEBPA, JAK2, IDH1, IDH2, KIT, KRAS, NRAS, NPM1, ASXL1, CBL, and MPL. On a median, two (range 0–32) somatic variants were detected per sample. Among all 91 reported cases, the most common somatically altered genes were RUNX1, TET2, ASXL1, BCOR, PHF6, and SRSF2, while variants in other genes were uniquely reported in specific malignancies. Remarkably, no additional acquired variants were detected in 21 (23%) of 91 samples. These cases were primarily associated with a pre-leukemic phenotype corroborating the notion that acquisition of somatic alterations is correlated with disease progression, particularly malignant transformation. The mutational signature of the four enrolled lymphoblastic HMs differed from myeloid samples as somatic variants were recurrently found in NOTCH1, PHF6, and TET2. Somatic RUNX1 alterations were found in 23 of all cases (25%) and in 23 of 53 patients (43%) with reported MDS, AML, or MDS/AML. None of the 30 non-HM patients had a secondary somatically acquired RUNX1 variant. RUNX1 being the most frequently investigated gene was also the most frequently mutated gene. This high frequency was significantly higher than in patients with assumed sporadic AML and is in line with data from previous reports, as somatic alterations of RUNX1 were reported as the most common somatic mutation in patients with RUNX1-FPD  . Nn = 53) with the non-HM group (n = 30) in our retrospective analyses  showed a somatic RUNX1 alteration, and 42 (79%) had additionally acquired variants  harbored any acquired variants besides RUNX1  providing growth advantage possibly leading to overt leukemia [RUNX1 alterations and, (ii) without acquired RUNX1 alterations. Thereby, we identified that in the group with acquired RUNX1 alterations, especially FLT3, BCOR, SRSF2, IDH1, and WT1 variants were frequently detected whereas recurrent variants in the group without acquired RUNX1 alteration were found in BCOR, NRAS, TET2, PHF6, and CDC25C. Previously, Brown et al. observed somatic variants affecting NRAS, SRSF2, DNMT3A and other genes associated with epigenetic regulation in RUNX1-FPD patients with AML [DNMT3A variants in four (11%) out of 37 RUNX1-FPD AMLs, one RUNX1-FPD myeloproliferative neoplasm, and one RUNX1-FPD thrombocytopenia patient. However, DNMT3A was not analyzed in seven of 37 RUNX1-FPD AML cases. Moreover, Brown et al. observed that somatic RUNX1 and somatic DNMT3A variants do not co-occur in RUNX1-FPD patients [RUNX1 and DNMT3A.We compared the groups of MDS, AML, and MDS/AML , although GATA2 gene was investigated in 76% of all 91 samples. In conclusion, our retrospective analyses do not support the hypotheses, that CDC25C and GATA2 are among the most frequently somatically affected genes in RUNX1-FPD.Somatic variants in families . Howeverhis data ,59,60, ahis data . A prevind GATA2 . In our TET2 occur in about 15–30% of patients with various sporadic myeloid malignancies [TET2 gene commonly occur biallelic in the context of sporadic hematologic neoplasms. Interestingly, we observed TET2 alterations in 15 out of 91 patients (16%). In one of the patients with cytopenia, two TET2 variants were identified. However, information was not given on whether these variants occurred in cis or in trans. TET2 variants were found in six MDS, AML and MDS/AML RUNX1-FPD cases . Therefore, TET2 variants in the analyzed RUNX1-FPD cohort appear to be less frequent than in sporadic leukemia. Since TET2 variants were concomitantly observed with variants in NPM1, FLT3, JAK2, RUNX1, CEBPA, CBL, and KRAS in sporadic AML patients [TET2-mutated AML samples. Thereby, we only found two acquired RUNX1 alterations and one CEBPA variant co-occurring in patients with TET2 variants. In sporadic HM, DNMT3A variants occur in about 20% of AML [TET2, JAK2, and SRSF2 occur in 10–60% of patients with sporadic chronic myelomonocytic leukemia [JAK2, an SRSF2, and an ASXL1 variant were detected in a patient with chronic myelomonocytic leukemia [CALR, MPL, and JAK2) are characteristic in the context of sporadic myeloproliferative neoplasms [JAK2 and a DNMT3A variant. Additionally, IDH1, FLT3, NPM1, and RASopathy-associated genes including NRAS, KRAS, and PTPN11 are frequently altered in sporadic HM [IDH1, FLT3 was the most frequently altered gene in the RUNX1-FPD AML subgroup (n = 37). However, no acquired variant was found in NPM1 [PTPN11 gene in 70 out of 91 samples (77%) revealed only one sample carrying a variant in this gene. Taken together, several frequently affected genes in sporadic HM are also affected by alterations in the analyzed retrospective RUNX1-FPD cohort. Besides different frequencies of additionally occurring genetic variants and the predominance of somatic RUNX1 variants, there are no characteristic differences between somatic variants detected in sporadic HM and RUNX1-FPD-associated HM.Next, we compared the identified somatic variants in RUNX1-FPD to those found in a patient with sporadic HM. Somatic mutations in gnancies ,62,63. Mpatients , we eval% of AML , 8% of M% of AML , and 17%% of AML . Variantleukemia . Here, aleukemia . Pathogeeoplasms ,70,71. Tradic HM ,73. DespOur retrospective analyses of somatic variants including 91 RUNX1-FPD cases had several limitations regarding sequencing panels, type and choice of samples, and differences in the provided information. Most of the literature focused on single genes or gene panels consisting of candidate genes already associated with HM leading to a data bias. Thus, it should be acknowledged that such approaches might overlook somatic variants in other genes not yet associated with HM. Noteworthy, variants with low variant allele fractions might also be sequencing artifacts. Moreover, it should be kept in mind that different tissues  investigated at different time points  might lead to divergent somatic signatures.RUNX1 mutations [DNMT3A, ASXL1, IDH2, and TET2 whereas secondary, driver mutations involve genes encoding several functional categories of proteins including transcription factors , signaling molecules , splicing factors , and proteins with other functions  [RUNX1 are usually secondary events, whereas, in FPD/AML, RUNX1 germline mutations are initiating or predisposing events [TET2, DNMT3A, and ASLX1 genes are also frequently detected in hematopoietic stem cells from non-diseased elderly individuals and are associated with age-related clonal hematopoiesis of indeterminate potential (CHIP) linked with an increased risk of HM and cardiovascular disease [RUNX1-mutated AML [DNMT3A and TET2, in 22% of preleukemic RUNX1-FPD patients and 40% of patients with myeloid malignancy [The diagnosis of RUNX1-FPD is of clinical interest for future management of the index patient and relatives at risk. It is currently not possible to predict an individual RUNX1-FPD patient’s risk of developing HM or the time of malignant transformation , becauseutations . Mutatio, SMC1A) ,76. In sg events . Of note disease ,78. Inteated AML . Moreovelignancy , which ilignancy .RUNX1 germline variant in order to assess baseline changes and exclude occult malignancy [FLT3, NPM1, DNMT3A, IDH1/2, and TET2, can be therapeutically targeted [https://www.genome.gov/Current-NHGRI-Clinical-Studies/hematologic-and-premalignant-conditions-associated-with-RUNX1-mutation, accessed on 7 June 2022).Monitoring RUNX1-FPD patients by sequencing panels to detect clonal hematopoiesis and/or alterations by variant allele fractions of already known variants may offer the opportunity to intervene at the pre-leukemic stage, prior to the appearance of overt MDS or frank leukemia. Previous data highlight the promise of surveillance and future potential for early intervention prior to the development of an overt HM in the at-risk population . Howeverlignancy . Followilignancy . Howevertargeted . PresentRUNX1 variants were clearly associated with MDS and/or AML in RUNX1-FPD patients and may serve as a genetic indicator of malignant transformation. The acquisition of different somatic variants may explain the clinical heterogeneity seen in RUNX1-FPD, even within affected families. However, the process of disease progression in RUNX1-FPD is complex since the pathogenesis cannot be explained merely by a single acquired variant. The spectrum of somatic variants and genes implicated in the progression is vast. It highlights the importance of somatic mutations in the development of frank leukemia. Recent advantages in NGS technologies reveal the entire picture of genetic alterations involved in tumorigenesis piece by piece. Applied panels should be adapted to cover all relevant genes. In addition, it is worth considering that not all acquired variants are necessarily attributed to disease progression and we need to distinguish between driver and passenger variants. Prospectively, synergistic effects of RUNX1 germline variants together with acquired variants should be evaluated with functional assays. Additionally, sharing identified germline and somatic variants with phenotypic information in appropriate databases in a standardized way, e.g., within the RUNX1db [Retrospective analysis supports the theory of stepwise malignant transformation in RUNX1-FPD. A correlation between the number of acquired variants and disease progression was identified. Moreover, somatic  RUNX1db  or the d