Construction of recombinant Lactococcus expressing thymosin and interferon fusion protein and its application as an immune adjuvant.

BACKGROUND: In recent years, biosafety and green food safety standards have increased the demand for immune enhancers and adjuvants. In the present study, recombinant food-grade Lactococcus lactis (r-L. lactis-Tα1-IFN) expressing thymosin Tα1 and chicken interferon fusion protein was constructed. RESULTS: The in vitro interactions with macrophages revealed a mixture of recombinant r-L. lactis-Tα1-IFN could significantly activate both macrophage J774-Dual™ NF-κB and interferon regulator (IRF) signaling pathways. In vitro interactions with chicken peripheral blood mononuclear cells (PBMCs) demonstrated that a mixture of recombinant r-L. lactis-Tα1-IFN significantly enhanced the expression levels of interferon (IFN)-γ, interleukin (IL)-10, CD80, and CD86 proteins in chicken PBMCs. Animal experiments displayed that injecting a lysis mixture of recombinant r-L. lactis-Tα1-IFN could significantly activate the proliferation of T cells and antigen-presenting cells in chicken PBMCs. Moreover, 16S analysis of intestinal microbiota demonstrated that injection of the lysis mixture of recombinant r-L. lactis-Tα1-IFN could significantly improve the structure and composition of chicken intestinal microbiota, with a significant increase in probiotic genera, such as Lactobacillus spp. Results of animal experiments using the lysis mixture of recombinant r-L. lactis-Tα1-IFN as an immune adjuvant for inactivated chicken Newcastle disease vaccine showed that the serum antibody titers of the experimental group were significantly higher than those of the vaccine control group, and the expression levels of cytokines IFN-γ and IL-2 were significantly higher than those of the vaccine control group. CONCLUSION: These results indicate that food-safe recombinant r-L. lactis-Tα1-IFN has potential as a vaccine immune booster and immune adjuvant. This study lays the foundation for the development of natural green novel animal immune booster or immune adjuvant.

Effect of prothymosin α on neuroplasticity following cerebral ischemia­reperfusion injury.

Prothymosin α (ProT), a highly acidic nuclear protein with multiple cellular functions, has shown potential neuroprotective properties attributed to its anti­necrotic and anti­apoptotic activities. The present study aimed to investigate the beneficial effect of ProT on neuroplasticity after ischemia­reperfusion injury and elucidate its underlying mechanism of action. Primary cortical neurons were either treated with ProT or overexpressing ProT by gene transfection and exposed to oxygen­glucose deprivation for 2 h in vitro. Immunofluorescence staining for ProT and MAP­2 was performed to quantify ProT protein expression and assess neuronal arborization. Mice treated with vehicle or ProT (100 µg/kg) and ProT overexpression in transgenic mice received middle cerebral artery occlusion for 50 min to evaluate the effect of ProT on neuroplasticity­associated protein following ischemia­reperfusion injury. The results demonstrated that in cultured neurons ProT significantly increased neurite lengths and the number of branches, accompanied by an upregulation mRNA level of brain­derived neurotrophic factor. Furthermore, ProT administration improved the protein expressions of synaptosomal­associated protein, 25 kDa and postsynaptic density protein 95 after ischemic­reperfusion injury in vivo. These findings suggested that ProT can potentially induce neuroplasticity effects following ischemia­reperfusion injury.

Ligustilide prevents thymic immune senescence by regulating Thymosin ß15-dependent spatial distribution of thymic epithelial cells.

BACKGROUND: Thymus is the most crucial organ connecting immunity and aging. The progressive senescence of thymic epithelial cells (TECs) leads to the involution of thymus under aging, chronic stress and other factors. Ligustilide (LIG) is a major active component of the anti-aging Chinese herbal medicine Angelica sinensis (Oliv.) Diels, but its role in preventing TEC-based thymic aging remains elusive. PURPOSE: This study explored the protective role of Ligustilide in alleviating ADM (adriamycin) -induced thymic immune senescence and its underlying molecular mechanisms. METHOD: The protective effect of Ligustilide on ADM-induced thymic atrophy was examined by mouse and organotypic models, and conformed by SA-ß-gal staining in TECs. The abnormal spatial distribution of TECs in the senescent thymus was analyzed using H&E, immunofluorescence and flow cytometry. The possible mechanisms of Ligustilide in ADM-induced thymic aging were elucidated by qPCR, fluorescence labeling and Western blot. The mechanism of Ligustilide was subsequently validated through actin polymerization inhibitor, genetic engineering to regulate Thymosin ß15 (Tß15) and Tß4 expression, molecular docking and ß Thymosin-G-actin cross-linking assay. RESULTS: At a 5 mg/kg dose, Ligustilide markedly ameliorated ADM-induced weight loss and limb grip weakness in mice. It also reversed thymic damage and restored positive selection impaired by ADM. In vitro, ADM disrupted thymic structure, reduced TECs number and hindered double negative (DN) T cell differentiation. Ligustilide counteracted these effects, promoted TEC proliferation and reticular differentiation, leading to an increase in CD4+ single positive (CD4SP) T cell proportion. Mechanistically, ADM diminished the microfilament quantity in immortalized TECs (iTECs), and lowered the expression of cytoskeletal marker proteins. Molecular docking and cross-linking assay revealed that Ligustilide inhibited the protein binding between G-actin and Tß15 by inhibiting the formation of the Tß15-G-actin complex, thus enhancing the microfilament assembly capacity in TECs. CONCLUSION: This study, for the first time, reveals that Ligustilide can attenuate actin depolymerization, protects TECs from ADM-induced acute aging by inhibiting the binding of Tß15 to G-actin, thereby improving thymic immune function. Moreover, it underscores the interesting role of Ligustilide in maintaining cytoskeletal assembly and network structure of TECs, offering a novel perspective for deeper understanding of anti thymic aging.

Therapeutic applications of thymosin peptides: a patent landscape 2018-present.

INTRODUCTION: Thymosins are small proteins found mainly in the thymus. They are involved in several biological processes, including immunoregulation, angiogenesis, and anti-inflammatory activity. Due to these multiple activities, thymosins are widely used as therapeutics. In fact, these peptides have shown interesting results in the treatment of eye disorders, anticancer therapy, and dysregulated immune disorders. AREA COVERED: We analyzed the thymosins therapeutic patent landscape describing the most significant patents published after 2018 and originally written in English, classified according to the different type of functions and diseases. We searched 'Thymosin' on Patentscope and Espacenet. EXPERT OPINION: Thymalfasin (Zadaxin) is the only FDA-approved thymosine-based drug used to treat chronic hepatitis B and C and as a chemotherapy inducer. This outcome demonstrates how thymosins can be exploited as therapeutics, especially in immunological and anti-cancer therapies. However, the development of modified thymosins could expand their therapeutic interest and application in different diseases. In fact, by chemical modifications, it is possible to increase proteolytic stability in the biological environment, enhance cell permeability, and stabilize the secondary structure of the peptide. Finally, the development of shorter sequences could reduce the cost and production time of these thymosin-based drugs.

Immune modulation via dendritic cells by the effect of Thymosin-alpha-1 on immune synapse in HCMV infection.

Tα1 (Thymosin-alpha-1) is a thymus-derived hormone that has been demonstrated to be effective on diverse immune cell subsets. The objective of this study was to determine the in vitro immunomodulatory effect of Tα1 in human cytomegalovirus (HCMV) infection. Dendritic cells (DCs) were isolated from peripheral blood mononuclear cells (PBMCs) by negative selection and cultured in the presence or absence of Tα1. The immunophenotyping of DCs was characterised by multiparametric flow cytometry assessing CD40, CD80, TIM-3 and PDL-1 markers, as well as intracellular TNFα production. Then, autologous CD4+ or CD8+ T-Lymphocytes (TLs) isolated by negative selection from PBMCs were co-cultured with DCs previously treated with Tα1 in the presence or absence of HCMV. Intracellular TNFα, IFNγ, IL-2 production, CD40-L and PD-1 expression were assessed through immunophenotyping, and polyfunctionality in total TLs and memory subsets were evaluated. The results showed that Tα1 increased CD40, CD80, TIM-3 and TNFα intracellular production while decreasing PDL-1 expression, particularly on plasmacytoid dendritic cells (pDCs). Therefore, Tα1 modulated the production of TNFα, IFNγ and IL-2 in both total and memory subsets of CD4+ and CD8+ TLs by upregulating CD40/CD40-L and downregulating PDL-1/PD-1 expression. Our study concludes that Tα1 enhances antigen-presenting capacity of DCs, improves TLs responses to HCMV infection, and enhances the polyfunctionality of CD8+ TLs. Consequently, Tα1 could be an alternative adjuvant for use in therapeutic cell therapy for immunocompromised patients.

The Clinical Relevance and Functional Implications of Thymosin Beta-10 in Glioma.

Glioma is a highly aggressive form of brain cancer characterized by limited treatment options and poor patient prognosis. In this study, we aimed to elucidate the oncogenic role of thymosin beta-10 (TMSB10) in glioma through comprehensive analyses of patient data from the TCGA and GTEx databases. Our investigation encompassed several key aspects, including the analysis of patients' clinical characteristics, survival analysis, in vitro and in vivo functional experiments, and the exploration of correlations between TMSB10 expression and immune cell infiltration. Our findings revealed a significant upregulation of TMSB10 expression in glioma tissues compared to normal brain tissues, with higher expression levels observed in tumors of advanced histological grades. Moreover, we observed positive correlations between TMSB10 expression and patient age, while no significant association with gender was detected. Additionally, TMSB10 exhibited marked elevation in gliomas with wild-type IDH and noncodeletion of 1p/19q. Survival analysis indicated that high TMSB10 expression was significantly associated with worse overall survival, disease-specific survival, and progression-free survival in glioma patients. Functionally, knockdown of TMSB10 in glioma cells resulted in reduced cellular growth rates and impaired tumor growth in xenograft models. Furthermore, our study revealed intriguing correlations between TMSB10 expression and immune cell infiltration within the tumor microenvironment. Specifically, TMSB10 showed negative associations with plasmacytoid dendritic cells (pDC) and γδ T cells (Tgd), while displaying positive correlations with neutrophils and macrophages. These findings collectively provide valuable insights into the oncogenic properties of TMSB10 in glioma, suggesting its potential as a therapeutic target and a biomarker for patient stratification.

Antioxioxidant and antiapoptotic effects of Thymosin ß4 in Aß-induced SH-SY5Y cells via the 5-HTR1A/ERK axis.

Alzheimer's disease (AD) is a common amnestic cognitive impairment characterised by ß-amyloid (Aß) plaques deposit in the brain of the elderly. AD is a yet incurable disease due to its unknown exact pathogenesis and unavailability of effective remedies in clinical application. Thymosin ß4 (Tß4) is a housekeeping protein that plays important role in cell proliferation, migration and differentiation. It has the ability to protect and repair neurons however it is still unclear involvement in AD. Therefore, the aim of this study is to elucidate the role and mechanism of Tß4 in mediating the improvement of AD. AD-like cell model was constructed in neuroblastoma cell line SH-SY5Y treated with Aß. Overexpression of Tß4 were done using lentivirus infection and downregulation through siRNA transfection. We performed western blot and flow cytometry to study the apoptosis and standard kits to measure the oxidative stress-associated biomarkers. There is significant increased in viability and decreased apoptosis in Tß4 overexpression group compared to control. Furthermore, overexpression of Tß4 suppressed the expression of pro-apoptotic markers such as Caspase-3, Caspase-8, and Bax meanwhile upregulated the expression of anti-apoptotic gene Bcl-2. Tß4 alleviated oxidative damage by reducing MDA, LDH and ROS and increasing SOD and GSH-PX in Aß-treated SH-SY5Y cells. We found that Tß4 inhibit ERK/p38 MAPK pathway and intensify the expression of 5-HTR1A. Additionally, we showed that upregulation of 5-HTR1A dampened the Tß4 to activate ERK signalling. In conclusion, our study revealed the neuroprotective role of Tß4 in AD which may open up new therapeutic applications in AD treatment.

Thymosin α1 modulated the immune landscape of COVID-19 patients revealed by single-cell RNA and TCR sequencing.

BACKGROUND: The Coronavirus disease-19 (COVID-19) pandemic has posed a serious threat to global health. Thymosin α1 (Tα1) was considered to be applied in COVID-19 therapy. However, the data remains limited. METHODS: Participants with or without Tα1 treatment were recruited. Single cell RNA-sequencing (scRNA-seq) and T cell receptor-sequencing (TCR-seq) of the peripheral blood mononuclear cell (PBMC) samples were done to analyze immune features. The differential expression analysis and functional enrichment analysis were performed to explore the mechanism of Tα1 therapy. RESULTS: 33 symptomatic SARS-CoV-2-infected individuals (COV) and 11 healthy controls (HC) were enrolled in this study. The proportion of CD3+ KLRD1+ NKT, TBX21+ CD8+ NKT was observed to increase in COVID-19 patients with Tα1 treatment (COVT) than those without Tα1 (COV) (p = 0.024; p = 0.010). These two clusters were also significantly higher in Health controls with Tα1 treatment (HCT) than those without Tα1 (HC) (p = 0.016; p = 0.031). Besides, a series of genes and pathways related to immune responses were significantly higher enriched in Tα1 groups TBX21+ CD8+ NKT, such as KLRB1, PRF1, natural killer cell-mediated cytotoxicity pathway, chemokine signaling pathway, JAK-STAT signaling pathway. The increased TRBV9-TRBJ1-1 pair existed in both HCs and COVID-19 patients after Tα1 treatment. 1389 common complementarity determining region 3 nucleotides (CDR 3 nt) were found in COV and HC, while 0 CDR 3 nt was common in COVT and HCT. CONCLUSIONS: Tα1 increased CD3+ KLRD1+ NKT, TBX21+ CD8+ NKT cell proportion and stimulated the diversity of TCR clones in COVT and HCT. And Tα1 could regulate the expression of genes associated with NKT activation or cytotoxicity to promote NKT cells. These data support the use of Tα1 in COVID-19 patients.

Detection and quantification of the metabolite Ac-Tß1-14 in in vitro experiments and urine of rats treated with Ac-Tß4: A potential biomarker of Ac-Tß4 for doping tests.

Thymosin ß4 (Tß4) was reported to exert various beneficial bioactivities such as tissue repair, anti-inflammation, and reduced scar formation, and it is listed on the prohibited substances in sports by the World Anti-Doping Agency. However, no metabolism studies of Tß4 were reported yet. Previously, our lab reported in in vitro experiment that a total of 13 metabolites were found by using multiple enzymes, and six metabolites (Ac-Tß31-43 , Ac-Tß17-43 , Ac-Tß1-11 , Ac-Tß1-14 , Ac-Tß1-15 , and Ac-Tß1-17 ) were confirmed by comparing with the synthetic standards. This study was aimed at identifying new metabolites of Tß4 leucine aminopeptidase (LAP), human kidney microsomes (HKM), cultured huvec cells, and rats after administration of Tß4 protein to develop biomarkers for detecting doping drugs in sports. A method for detecting and quantifying Ac-Tß1-14 was developed and validated using Q-Exactive orbitrap mass spectrometry. The limit of detection (LOD) and limit of quantification (LOQ) of the Ac-Tß1-14 were 0.19 and 0.58 ng/mL, respectively, and showed a good linearity (r2 = 0.9998). As a result, among the six metabolites above, Ac-Tß1-14 , as a common metabolite, was found in LAP, HKM, huvec cells exposed to Tß4, and the urine of rats intraperitoneally treated with 20-mg/kg Tß4. And the metabolite Ac-Tß1-14 was quantitatively determined by 48 h in rats, with the highest concentration occurring between 0 and 6 h. Ac-Tß1-14 was not detected in non-treated control groups, including human blank urine. These results suggest that Ac-Tß1-14 in urine is a potential biomarker for screening the parent Tß4 in doping tests.

Targeted Deletion of Thymosin Beta 4 in Hepatic Stellate Cells Ameliorates Liver Fibrosis in a Transgenic Mouse Model.

Liver fibrosis is the most common feature of liver disease, and activated hepatic stellate cells (HSCs) are the main contributors to liver fibrosis. Thus, finding key targets that modulate HSC activation is important to prevent liver fibrosis. Previously, we showed that thymosin ß4 (Tß4) influenced HSC activation by interacting with the Hedgehog pathway in vitro. Herein, we generated Tß4 conditional knockout (Tß4-flox) mice to investigate in vivo functions of Tß4 in liver fibrosis. To selectively delete Tß4 in activated HSCs, double-transgenic (DTG) mice were generated by mating Tß4-flox mice with α-smooth muscle actin (α-Sma)-Cre-ERT2 mice, and these mice were administered carbon tetrachloride (CCl4) or underwent bile duct ligation to induce liver fibrosis. Tß4 was selectively suppressed in the activated HSCs of DTG mouse liver, and this reduction attenuated liver injury, including fibrosis, in both fibrotic models by repressing Hedgehog (Hh) signaling. In addition, the re-expression of Tß4 by an adeno-associated virus reversed the effect of HSC-specific Tß4 deletion and led to liver fibrosis with Hh activation in CCl4-exposed mice treated with tamoxifen. In conclusion, our results demonstrate that Tß4 is a crucial regulator of HSC activation, suggesting it as a novel therapeutic target for curing liver fibrosis.

Provocative non-canonical roles of p53 and AKT signaling: A role for Thymosin ß4 in medulloblastoma.

The PI3K/AKT and p53 pathways are key regulators of cancer cell survival and death, respectively. Contrary to their generally accepted roles, several lines of evidence, including ours in medulloblastoma, the most common childhood brain cancer, highlight non-canonical functions for both proteins and show a complex context-dependent dynamic behavior in determining cell fate. Interestingly, p53-mediated cell survival and AKT-mediated cell death can dominate in certain conditions, and these interchangeable physiological functions may potentially be manipulated for better clinical outcomes. This review article presents studies in which p53 and AKT behave contrary to their well-established functions. We discuss the factors and circumstances that may be involved in mediating these changes and the implications of these unique roles of p53 and AKT in devising therapeutic strategies. Lastly, based on our recent finding of Thymosin beta 4-mediated chemosensitivity via an AKT-p53 interaction in medulloblastoma cells, we also discuss the possible implications of Thymosin beta-4 in enhancing drug sensitivity in this deadly childhood disease.

Cell starvation increases uptake of extracellular Thymosin ß4 and its complexes with calcium.

Cell metastasis is the main cause of cancer mortality. Inhibiting early events during cell metastasis and invasion could significantly improve cancer prognosis, but the initial mechanisms of cell transition and migration are barely known. Calcium regulates cell migration, whilst Thymosin ß4 is a G-actin and iron binding peptide associated with tumor metastasis and ferroptosis. Under normal cell growth conditions, intracellular free calcium ions and Thymosin ß4 concentrations are strictly regulated, and are not influenced by extracellular supplementation. However, cell starvation decreases intracellular Thymosin ß4 and increases extracellular peptide uptake above the normal range. Unexpectedly, cell starvation significantly increases internalization of extracellular Ca2+/Thymosin ß4 complexes. Elucidating the role of Ca2+/Thymosin ß4 in the early events of metastasis will likely be important in the future to develop therapies targeting metastasis.

Thymosin beta-4 denotes new directions towards developing prosperous anti-aging regenerative therapies.

Our dream of defeating the processes of organ damage and aging remains a challenge scientists pursued for hundreds of years. Although the goal is to successfully treat the body as a whole, steps towards regenerating individual organs are even considered significant. Since initial approaches utilizing only progenitor cells appear limited, we propose interconnecting our collective knowledge regarding aging and embryonic development may lead to the discovery of molecules which provide alternatives to effectively reverse cellular damage. In this review, we introduce and summarize our results regarding Thymosin beta-4 (TB4) to support our hypothesis using the heart as model system. Accordingly, we investigated the developmental expression of TB4 in mouse embryos and determined the impact of the molecule in adult animals by systemically injecting the peptide following acute cardiac infarction or with no injury. Our results proved, TB4 is expressed in the developing heart and promotes cardiac cell migration and survival. In adults, the peptide enhances myocyte survival and improves cardiac function after coronary artery ligation. Moreover, intravenous injections of TB4 alter the morphology of the adult epicardium, and the changes resemble the characteristics of the embryo. Reactivation of the embryonic program became equally reflected by the increased number of cardiac vessels and by the alteration of the gene expression profile typical of the embryonic state. Moreover, we discovered TB4 is capable of epicardial progenitor activation, and revealed the effect is independent of hypoxic injury. By observing the above results, we believe, further discoveries and consequential postnatal administration of developmentally relevant candidate molecules such as TB4 may likely result in reversing aging processes and accelerate organ regeneration in the human body.

Thymosin ß4 and prothymosin α promote cardiac regeneration post-ischaemic injury in mice.

AIMS: The adult mammalian heart is a post-mitotic organ. Even in response to necrotic injuries, where regeneration would be essential to reinstate cardiac structure and function, only a minor percentage of cardiomyocytes undergo cytokinesis. The gene programme that promotes cell division within this population of cardiomyocytes is not fully understood. In this study, we aimed to determine the gene expression profile of proliferating adult cardiomyocytes in the mammalian heart after myocardial ischaemia, to identify factors to can promote cardiac regeneration. METHODS AND RESULTS: Here, we demonstrate increased 5-ethynyl-2'deoxyuridine incorporation in cardiomyocytes 3 days post-myocardial infarction in mice. By applying multi-colour lineage tracing, we show that this is paralleled by clonal expansion of cardiomyocytes in the borderzone of the infarcted tissue. Bioinformatic analysis of single-cell RNA sequencing data from cardiomyocytes at 3 days post ischaemic injury revealed a distinct transcriptional profile in cardiomyocytes expressing cell cycle markers. Combinatorial overexpression of the enriched genes within this population in neonatal rat cardiomyocytes and mice at postnatal day 12 (P12) unveiled key genes that promoted increased cardiomyocyte proliferation. Therapeutic delivery of these gene cocktails into the myocardial wall after ischaemic injury demonstrated that a combination of thymosin beta 4 (TMSB4) and prothymosin alpha (PTMA) provide a permissive environment for cardiomyocyte proliferation and thereby attenuated cardiac dysfunction. CONCLUSION: This study reveals the transcriptional profile of proliferating cardiomyocytes in the ischaemic heart and shows that overexpression of the two identified factors, TMSB4 and PTMA, can promote cardiac regeneration. This work indicates that in addition to activating cardiomyocyte proliferation, a supportive environment is a key for regeneration to occur.

Hypermethylation of thymosin ß4 predicts a poor prognosis for patients with acute-on-chronic hepatitis B liver failure.

BACKGROUND: It has been demonstrated that thymosin ß4 (Tß4) could inflect the severity of acute-on-chronic hepatitis B liver failure (ACHBLF), but the relationship between its methylation status and the prognosis of liver failure is not clear. This study aimed to determine Tß4 promoter methylation status in patients with ACHBLF and to evaluate its prognostic value. METHODS: The study recruited 115 patients with ACHBLF, 80 with acute-on-chronic hepatitis B pre-liver failure (pre-ACHBLF), and 86 with chronic hepatitis B (CHB). In addition, there were 36 healthy controls (HCs) from the Department of Hepatology, Qilu Hospital of Shandong University. The 115 patients with ACHBLF were divided into three subgroups: 33 with early stage ACHBLF (E-ACHBLF), 42 with mid-stage ACHBLF (M-ACHBLF), and 40 with advanced stage ACHBLF (A-ACHBLF). Tß4 promoter methylation status in peripheral blood mononuclear cells (PBMCs) was measured by methylation-specific polymerase chain reaction, and mRNA was detected by quantitative real-time polymerase chain reaction. RESULTS: Methylation frequency of Tß4 was significantly higher in patients with ACHBLF than in those with pre-ACHBLF, CHB or HCs. However, expression of Tß4 mRNA showed the opposite trend. In patients with ACHBLF, Tß4 promoter methylation status correlated negatively with mRNA levels. The 3-month mortality of ACHBLF in the methylated group was significantly higher than that in the unmethylated group. Also, Tß4 promoter methylation frequency was lower in survivors than in non-survivors. When used to predict the 1-, 2-, and 3-month incidence of ACHBLF, Tß4 methylation status was better than the model for end-stage liver disease (MELD) score. The predictive value of Tß4 methylation was higher than that of MELD score for the mortality of patients with E-ACHBLF and M-ACHBLF, but not for A-ACHBLF. CONCLUSIONS: Tß4 methylation might be an important early marker for predicting disease incidence and prognosis in patients with ACHBLF.

Thymosin ß4 and the anti-fibrotic switch

Wound healing involves a rapid response to the injury by circulating cells, followed by inflammation with an influx of inflammatory cells that release various factors. Soon after, cellular proliferation begins to replace the damaged cells and extracellular matrix, and then tissue remodeling restores normal tissue function. Various factors can lead to pathological wound healing when excessive and irreversible connective tissue/extracellular matrix deposition occurs, resulting in fibrosis. The process is initiated when immune cells, such as macrophages, release soluble factors that stimulate fibroblasts. TGFß is the most well-characterized macrophage derived pro-fibrotic mediator. Other soluble mediators of fibrosis include connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF), and interleukin 10 (IL-10). Thymosin ß4 (Tß4) has shown therapeutic benefit in preventing fibrosis/scarring in various animal models of fibrosis/scarring. The mechanism of action of Tß4 appears related, in part, to a reduction in the inflammatory response, including a reduction in macrophage infiltration, decreased levels of TGFß and IL-10, and reduced CTGF activation, resulting in both prevention of fibroblast conversion to myofibroblasts and production of normally aligned collagen fibers. The amino N-terminal end of Tß4, SDKP (serine-aspartate-lysine-proline), appears to contain the majority of anti-fibrotic activity and has shown excellent efficacy in many animal models of fibrosis, including liver, lung, heart, and kidney fibrosis. Ac-SDKP not only prevents fibrosis but can reverse fibrosis. Unanswered questions and future directions will be presented with regard to therapeutic uses alone and in combination with already approved drugs for fibrosis.

Thymosin ß4 and Actin: Binding Modes, Biological Functions and Clinical Applications.

Thymosin ß4 (Tß4) is the ß-thymosin (Tßs) with the highest expression level in human cells; it makes up roughly 70-80% of all Tßs in the human body. Combining the mechanism and activity studies of Tß4 in recent years, we provide an overview of the subtle molecular mechanism, pharmacological action, and clinical applications of Tß4. As a G-actin isolator, Tß4 inhibits the polymerization of G-actin by binding to the matching site of G-actin in a 1:1 ratio through conformational and spatial effects. Tß4 can control the threshold concentration of G-actin in the cytoplasm, influence the balance of depolymerization and polymerization of F-actin (also called Tread Milling of F-actin), and subsequently affect cell's various physiological activities, especially motility, development and differentiation. Based on this, Tß4 is known to have a wide range of effects, including regulation of inflammation and tumor metastasis, promotion of angiogenesis, wound healing, regeneration of hair follicles, promotion of the development of the nervous system, and improving bone formation and tooth growth. Tß4 therefore has extensive medicinal applications in many fields, and serves to preserve the kidney, liver, heart, brain, intestine, and other organs, as well as hair loss, skin trauma, cornea repairing, and other conditions. In this review, we focus on the mechanism of action and clinical application of Tß4 for its main biological functions.

The Role of Tß4-POP-Ac-SDKP Axis in Organ Fibrosis.

Fibrosis is a pathological process in which parenchymal cells are necrotic and excess extracellular matrix (ECM) is accumulated due to dysregulation of tissue injury repair. Thymosin ß4 (Tß4) is a 43 amino acid multifunctional polypeptide that is involved in wound healing. Prolyl oligopeptidase (POP) is the main enzyme that hydrolyzes Tß4 to produce its derivative N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) which is found to play a role in the regulation of fibrosis. Accumulating evidence suggests that the Tß4-POP-Ac-SDKP axis widely exists in various tissues and organs including the liver, kidney, heart, and lung, and participates in the process of fibrogenesis. Herein, we aim to elucidate the role of Tß4-POP-Ac-SDKP axis in hepatic fibrosis, renal fibrosis, cardiac fibrosis, and pulmonary fibrosis, as well as the underlying mechanisms. Based on this, we attempted to provide novel therapeutic strategies for the regulation of tissue damage repair and anti-fibrosis therapy. The Tß4-POP-Ac-SDKP axis exerts protective effects against organ fibrosis. It is promising that appropriate dosing regimens that rely on this axis could serve as a new therapeutic strategy for alleviating organ fibrosis in the early and late stages.

Nuclear factor (erythroid-derived 2)-like 2 counter-regulates thymosin beta-4 expression and primary cilium formation for HeLa cervical cancer cell survival.

We investigated the function of thymosin beta-4 (TB4) expression and primary cilium (PC) formation via the underlying Nrf2-dependent mechanism for cervical cancer cell (CC) survival under conditions of serum deprivation (SD). TB4 silencing was achieved using RNA interference. The percentage of PC formation was analyzed by immunofluorescence staining. Nrf2 expression was modified by the preparation of stable Nrf2-knockdown cells with shNrf2 and the overexpression of Nrf2 with pcDNA-Nrf2 plasmids. Gene expression was measured using reverse-transcription PCR, Gaussia luciferase assay, and western blotting. Cell viability was assessed using the MTT assay or CellTiter Glo assay. Reactive oxygen species (ROS) were detected with flow cytometry. CCs incubated in SD without fetal bovine serum remained viable, and SD increased PC formation and TB4 transcription. CC viability was further decreased by treatment with ciliobrevin A to inhibit PC formation or TB4-siRNA. SD increased ROS, including H2O2. N-acetylcysteine inhibited ROS production following H2O2 treatment or SD, which also decreased PC formation and TB4 transcription. Meanwhile, H2O2 increased PC formation, which was attenuated in response to TB4 siRNA. Treatment with H2O2 increased Nrf2 expression, antioxidant responsive element (ARE) activity, and PC formation, which were inhibited by the Nrf2 inhibitor clobestasol propionate. Nrf2 knockdown via expression of Tet-On shNrf2 enhanced ROS production, leading to increased PC formation and decreased TB4 expression; these effects were counteracted by Nrf2 overexpression. Our data demonstrate that Nrf2 counter-regulates TB4 expression and PC formation for CC survival under conditions of SD, suggesting cervical CC survival could be upregulated by PC formation via Nrf2 activation and TB4 expression.

First Evidence of the Expression and Localization of Prothymosin α in Human Testis and Its Involvement in Testicular Cancers.

Prothymosin α (PTMA) is a phylogenetically conserved polypeptide in male gonads of Vertebrates. In Mammals, it is a ubiquitous protein, and, possessing a random-coil structure, it interacts with many other partners, in both cytoplasmic and nuclear compartments. PTMA has been widely studied during cell progression in different types of cancer because of its anti-apoptotic and proliferative properties. Here, we provided the first evidence of PTMA expression and localization in human testis and in two testicular cancers (TC): classic seminoma (CS) and Leydig cell tumor (LCT). Data showed that its protein level, together with that of proliferating cell nuclear antigen (PCNA), a cell cycle progression marker, increased in both CS and LCT samples, as compared to non-pathological (NP) tissue. Moreover, in the two-cancer tissue, a decreased apoptotic rate and an increased autophagic flux was also evidenced. Results confirmed the anti-apoptotic action of PTMA, also suggesting that it can act as a switcher from apoptosis to autophagy, to favor the survival of testicular cancer cells when they develop in adverse environments. Finally, the combined data, even if they need to be further validated, add new insight into the role of PTMA in human normal and pathological testicular tissue.