We showed earlier that human beta-thymosin 15 (Tb15) is up-regulated in prostate cancer, confirming studies from others that propagated Tb15 as a prostate cancer biomarker. In this first report on mouse Tb15, we show that, unlike in humans, four Tb15-like isoforms are present in mouse. We used phylogenetic analysis of deuterostome beta-thymosins to show that these four new isoforms cluster within the vertebrate Tb15-clade. Intriguingly, one of these mouse beta-thymosins, Tb15r, consists of two beta-thymosin domains. The existence of such a repeat beta-thymosin is so far unique in vertebrates, though common in lower eukaryotes. Biochemical data indicate that Tb15r potently sequesters actin. In a cellular context, Tb15r behaves as a bona fide beta-thymosin, lowering central stress fibre content. We reveal that a complex genomic organization underlies Tb15r expression: Tb15r results from read-through transcription and alternative splicing of two tandem duplicated mouse Tb15 genes. Transcript profiling of all mouse beta-thymosin isoforms (Tb15s, Tb4 and Tb10) reveals that two isoform switches occur between embryonic and adult tissues, and indicates Tb15r as the major mouse Tb15 isoform in adult cells. Tb15r is present also in mouse prostate cancer cell lines. This insight into the mouse Tb15 family is fundamental for future studies on Tb15 in mouse (prostate) cancer models.

Thymosin beta-4 (Tβ4) is known to induce hair growth and hair follicle (HF) development; however, its mechanism of action is unknown. We generated mice that overexpressed Tβ4 in the epidermis, as well as Tβ4 global knockout mice, to study the role of Tβ4 in HF development and explore the mechanism of Tβ4 on hair growth. To study Tβ4 function, we depilated control and experimental mice and made tissue sections stained with hematoxylin and eosin (H&E). To explore the effect of Tβ4 on hair growth and HF development, the mRNA and protein levels of Tβ4 and VEGF were detected by real-time PCR and western blotting in control and experimental mice. Protein expression levels and the phosphorylation of P38, ERK and AKT were also examined by western blotting. The results of depilation indicated that hair re-growth was faster in Tβ4-overexpressing mice, but slower in knockout mice. Histological examination revealed that Tβ4-overexpressing mice had a higher number of hair shafts and HFs clustered together to form groups, while the HFs of control mice and knockout mice were separate. Hair shafts in knockout mice were significantly reduced in number compared with control mice. Increased Tβ4 expression at the mRNA and protein levels was confirmed in Tβ4-overexpressing mice, which also had increased VEGF expression. On the other hand, knockout mice had reduced levels of VEGF expression. Mechanistically, Tβ4-overexpressing mice showed increased protein expression levels and phosphorylation of P38, ERK and AKT, whereas knockout mice had decreased levels of both expression and phosphorylation of these proteins. Tβ4 appears to regulate P38/ERK/AKT signaling via its effect on VEGF expression, with a resultant effect on the speed of hair growth, the pattern of HFs and the number of hair shafts.

The sheared avian intestinal villus-crypts exhibit high tendency to self-repair and develop enteroids in culture. Presuming that this transition process involves differential biomolecular changes, we employed matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) to find whether there were differences in the spectral profiles of sheared villi versus the enteroids, assessed in the mass range of 2-18 kDa. The results showed substantial differences in the intensities of the spectral peaks, one particularly corresponding to the mass of 4963 Da, which was significantly low in the sheared villus-crypts compared with the enteroids. Based on our previous results with other avian tissues and further molecular characterization by LC-ESI-IT-TOF-MS, and multiple reaction monitoring (MRM), the peak was identified to be thymosin β4 (Tβ4), a ubiquitously occurring regulatory peptide implicated in wound healing process. The identity of the peptide was further confirmed by immunohistochemistry which showed it to be present in a very low levels in the sheared villi but replete in the enteroids. Since Tβ4 sequesters G-actin preventing its polymerization to F-actin, we compared the changes in F-actin by its immunohistochemical localization that showed no significant differences between the sheared villi and enteroids. We propose that depletion of Tβ4 likely precedes villous reparation process. The possible mechanism for the differences in Tβ4 profile in relation to the healing of the villus-crypts to developing enteroids is discussed.

Glomerular disease is characterized by morphologic changes in podocyte cells accompanied by inflammation and fibrosis. Thymosin β4 regulates cell morphology, inflammation, and fibrosis in several organs and administration of exogenous thymosin β4 improves animal models of unilateral ureteral obstruction and diabetic nephropathy. However, the role of endogenous thymosin β4 in the kidney is unknown. We demonstrate that thymosin β4 is expressed prominently in podocytes of developing and adult mouse glomeruli. Global loss of thymosin β4 did not affect healthy glomeruli, but accelerated the severity of immune-mediated nephrotoxic nephritis with worse renal function, periglomerular inflammation, and fibrosis. Lack of thymosin β4 in nephrotoxic nephritis led to the redistribution of podocytes from the glomerular tuft toward the Bowman capsule suggesting a role for thymosin β4 in the migration of these cells. Thymosin β4 knockdown in cultured podocytes also increased migration in a wound-healing assay, accompanied by F-actin rearrangement and increased RhoA activity. We propose that endogenous thymosin β4 is a modifier of glomerular injury, likely having a protective role acting as a brake to slow disease progression.

The advent of immune checkpoint inhibitors has represented a major boost in cancer therapy, but safety concerns are increasingly being recognized. Indeed, although beneficial at the tumor site, unlocking a safeguard mechanism of the immune response may trigger autoimmune-like effects at the periphery, thus making the safety of immune checkpoint inhibitors a research priority. Herein, we demonstrate that thymosin α1 (Tα1), an endogenous peptide with immunomodulatory activities, can protect mice from intestinal toxicity in a murine model of immune checkpoint inhibitor-induced colitis. Specifically, Tα1 efficiently prevented immune adverse pathology in the gut by promoting the indoleamine 2,3-dioxygenase (IDO) 1-dependent tolerogenic immune pathway. Notably, Tα1 did not induce IDO1 in the tumor microenvironment, but rather modulated the infiltration of T-cell subsets by inverting the ratio between CD8+ and Treg cells, an effect that may depend on Tα1 ability to regulate the differentiation and chemokine expression profile of DCs. Thus, through distinct mechanisms that are contingent upon the context, Tα1 represents a plausible candidate to improve the safety/efficacy profile of immune checkpoint inhibitors.

A peptide with a sequence identical to rat thymosin beta(Tb)15 was reported to be upregulated in human prostate cancer. However, in this report we provide evidence that TbNB, initially identified in human neuroblastoma, is the only Tb isoform upregulated in human prostate cancer and that the Tb15 sequence is not present herein. In addition, we demonstrate that human TbNB has a higher affinity for actin in comparison to Tb4 and promotes cell migration. In combination, this experimentally validates TbNB as functional homologue of rat Tb15 in the human organism and clarifies the current composition of the human Tb family.

Induced pluripotent stem cells (iPSC) constitute a powerful tool to study cardiac physiology and represents a promising treatment strategy to tackle cardiac disease. However, iPSCs remain relatively immature after differentiation. Additionally, engineered heart tissue (EHT) has been investigated as a therapy option in preclinical disease models with promising results, although their vascularization and functionality leave room for improvement. Thymosin β4 (Tβ4) has been shown to promote the differentiation of progenitor cell lines to cardiomyocytes while it also induces angiogenic sprouting and vascular maturation. We examined the potential impact of Tβ4 to enhance maturation of cardiomyocytes from iPSCs. Assessing the expression of transcription factors associated with cardiac differentiation, we were able to demonstrate the increased generation of cells displaying cardiomyocyte characteristics in vitro. Furthermore, we demonstrated, in a zebrafish model of embryonic vascular development, that Tβ4 is crucial for the proper execution of lymphatic and angiogenic vessel sprouting. Finally, utilizing Tβ4-transduced EHTs generated from mice genetically engineered to label endothelial cells in vitro, we show that treatment with Tβ4 promotes vascularization and contractility in EHTs, highlighting Tβ4 as a growth factor improving the formation of cardiomyocytes from iPSC and enhancing the performance of EHTs generated from neonatal cardiomyocytes.

Thymosin β-4(Tβ-4) is a macromolecular protein drug with potential for drug development in wound repair but is limited by the shortcomings of macromolecular protein, such as large volumes, poor membrane permeability, and unstable physicochemical characteristics. Ethosomes could enhance cell membrane fluidity and reduce epidermal membrane density to make macromolecular drugs through the stratum corneum into the deeper layers of the skin easily. Herein, we developed and characterized a novel transdermal delivery vehicle to load macromolecular protein peptides and use Tβ-4 as a model drug wrapped into ethosomes.

Glioblastomas are high-grade, malignant CNS neoplasms that are nearly always fatal within 12 months of diagnosis. Immunotherapy using proinflammatory cytokines such as IL-2 or IL-12 may prolong survival with glioblastoma. Thymosin-alpha1 (Talpha1) is a thymic hormone and immunemodulator that increase IL-2 production and T-cell proliferation. We examined potential therapeutic effects of Talpha1 in experimental in vivo glioblastoma, and characterized Talpha1's anti-tumor effects in vitro. Rar 9L cells (10(4)) were implanted into the right frontal lobe of adult Long Evans rats that were subsequently treated with vehicle, BCNU, Talpha1, or Talpha1+BCNU from postoperative day 6. Talpha1+BCNU significantly lowered tumor burdens, and increased cure rates. In vitro experiments demonstrated that Talpha1 had no direct effect on viability or mitochondrial function, and instead, it increased expression of pro-apoptosis genes, including FasL, FasR and TNFalpha-R1 (65.89%, 44.08%, and 22.18%, resp.), and increased 9L cell sensitivity to oxidative stress. Moreover, Talpha1 enhanced 9L cell sensitivity to both Granzyme B- and BCNU-mediated killing. The findings suggest that Talpha1 enhances BCNUmediated eradication of glioblastoma in vivo, and that Talpha1 mediates its effects by activating pro-apoptosis mechanisms, rendering neoplastic cells more sensitive to oxidative stress and immune-mediated killing by Granzyme B and chemotherapeutic agents.

Human thymosin alpha 1 (Talpha1) is an important peptide in the development and senescence of immunological competence in human, and many studies have reported the expression of this peptide. In this study, we designed and synthesized the Talpha1 gene according to the E. coli codon usage preference and constructed a 6xTalpha1 concatemer. The latter was inserted into an E. coli expression vector pET-22b (+), and transformed into E. coli BL21 (DE3). After induction with IPTG, the concatemer protein was successfully expressed in E. coli then cleaved by hydroxylamine to release the Talpha1 monomer. Gly-SDS-PAGE and mass spectrometry confirmed that the recombinant protein was cleaved as intended. The bioactivity of the Talpha1 monomer was analyzed by lymphocyte proliferation and by mitochondrial activity in two different tumor cell lines. This study provides a description of the preparation of a bioactive Talpha1, which may prove useful in future biomedical research.

The thymus is the most sensitive organ under various pathophysiological conditions, such as aging, starvation, and infection. As a key stromal cell for T cell development, it is well-known that thymic epithelial cells (TECs) play an important role in the thymus response to the external environment. Thymosin beta 15 (Tβ15) is a G-actin binding protein secreted by TECs, it plays an important role in maintaining the dynamic balance of actin, angiogenesis, axonal formation, and wound healing, but the relationship between Tβ15 and TECs is not clear yet. Here, we show the impact of Tβ15 on the TEC's spatial development, as well as the T-cell differentiation and thymic output. As a result, TEC is the main effector cell of Tβ15 in the thymus. Tβ15 OX inhibits the chemotaxis of TECs to the medulla and subsequently blocks the positive selection of thymocytes from CD3+TCRβ+CD4+CD8+ double positive cells to CD3+TCRβ+CD4+CD8- single-positive (CD4SP) cells. Tβ15-knockdown accelerates the reticular differentiation of astral TECs and medullary TECs. Importantly, mice implanted with Tβ15-knockdown iTECs show high thymic output but low peripheral T cell maturity and activity. In a word, our results explain the role of Tβ15 on the differentiation and function of TECs and provide a new perspective for understanding the process of thymus development and degeneration.

We investigated the therapeutic effect of recombinant thymosin β4 (rTβ4) on rabbit autoimmune dacryoadenitis, an animal model of SS dry eye, and explore its mechanisms.

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the gene encoding for the ion channel Cystic Fibrosis Transmembrane conductance Regulator (CFTR). Long considered a lung disease for the devastating impact on the respiratory function, the recent diagnostic and therapeutic advances have shed the light on the extra-pulmonary manifestations of CF, including gastrointestinal, hepatobiliary and pancreatic symptoms. We have previously demonstrated that thymosin alpha1 (Tα1), a naturally occurring immunomodulatory peptide, displays multi-sided beneficial effects in CF that concur in ameliorating the lung inflammatory pathology. In the present study, by resorting to murine models of gut inflammation with clinical relevance for CF patients, we demonstrate that Tα1 can also have beneficial effects in extrapulmonary pathology. Specifically, Tα1 restored barrier integrity and immune homeostasis in the inflamed gut of CF mice as well as in mice with the metabolic syndrome, a disorder that may arise in CF patients with high caloric intake despite pancreatic sufficiency. The protective effects of Tα1 also extended to pancreas and liver, further emphasizing the beneficial effects of Tα1 in extra-pulmonary complications of CF. By performing wide-ranging multi-organ anti-inflammatory effects, Tα1 could potentially integrate current therapeutic approaches to tackle the complex symptomatology of CF disease.

Coronaviruses (CoVs) are enveloped and harbor an unusually large (30-32 kb) positive-strand linear RNA genome. Highly pathogenic coronaviruses cause severe acute respiratory syndrome (SARS) (SARS-CoV and SARS-CoV-2) and Middle East respiratory syndrome (MERS) (MERS-CoV) in humans. The coronavirus mouse hepatitis virus (MHV) infects mice and serves as an ideal model of viral pathogenesis, mainly because experiments can be conducted using animal-biosafety level-2 (A-BSL2) containment. Human thymosin beta-4 (Tβ4), a 43-residue peptide with an acetylated N-terminus, is widely expressed in human tissues. Tβ4 regulates actin polymerization and functions as an anti-inflammatory molecule and an antioxidant as well as a promoter of wound healing and angiogenesis. These activities led us to test whether Tβ4 serves to treat coronavirus infections of humans. To test this possibility, here, we established a BALB/c mouse model of coronavirus infection using mouse CoV MHV-A59 to evaluate the potential protective effect of recombinant human Tβ4 (rhTβ4). Such a system can be employed under A-BSL2 containment instead of A-BSL3 that is required to study coronaviruses infectious for humans. We found that rhTβ4 significantly increased the survival rate of mice infected with MHV-A59 through inhibiting virus replication, balancing the host's immune response, alleviating pathological damage, and promoting repair of the liver. These results will serve as the basis for further application of rhTβ4 to the treatment of human CoV diseases such as COVID-19.

To investigate the protective effect of a recombinant adeno-associated virus carrying thymosin β4 (AAV-Tβ4) on murine colitis via intracolonic administration.

The bHLH transcription factor Hand1 (Heart and neural crest-derived transcript-1) has a fundamental role in cardiovascular development; however, the molecular mechanisms have not been elucidated. In this paper we identify Thymosin β4 (Tβ4/Tmsb4x), which encodes an actin monomer-binding protein implicated in cell migration and angiogenesis, as a direct target of Hand1. We demonstrate that Hand1 binds an upstream regulatory region proximal to the promoter of Tβ4 at consensus Thing1 and E-Box sites and identify both activation and repression of Tβ4 by Hand1, through direct binding within either non-canonical or canonical E-boxes, providing new insight into gene regulation by bHLH transcription factors. Hand1-mediated activation of Tβ4 is essential for yolk sac vasculogenesis and embryonic survival, and administration of synthetic TB4 partially rescues yolk sac capillary plexus formation in Hand1-null embryos. Thus, we identify an in vivo downstream target of Hand1 and reveal impaired yolk sac vasculogenesis as a primary cause of early embryonic lethality following loss of this critical bHLH factor.

Thymosin β4 (Tβ4) has many physiological functions that are highly relevant to spinal cord injury (SCI), including neuronal survival, anti-inflammation, wound repair promotion, and angiogenesis. The present study investigated the therapeutic value of Tβ4 in SCI, with a focus on its neuroprotective, anti-inflammatory, and vasculoprotective properties. Tβ4 or a saline control was administered by intraperitoneal injection 30 min, 3 days, or 5 days after SCI with mild compression in rat. Locomotor recovery was tested with the Basso-Beattie-Bresnahan scale and a footprint analysis. All behavioral assessments were markedly improved with Tβ4 treatment. Histological examination at 7 days post injury showed that the numbers of surviving neurons and oligodendrocytes were significantly increased in Tβ4-treated animals compared to saline-treated controls. Levels of myelin basic protein, a marker of mature oligodendrocytes, in Tβ4-treated rats were 57.8% greater than those in saline-treated controls. The expression of ED1, a marker of activated microglia/macrophages, was reduced by 36.9% in the Tβ4-treated group compared to that of the saline-treated group. Tβ4 treatment after SCI was also associated with a significant decrease in pro-inflammatory cytokine gene expression and a significant increase in the mRNA levels of IL-10 compared to the control. Moreover, the size of lesion cavity delineated by astrocyte scar in the injured spinal cord was markedly reduced in Tβ4-treated animals compared to saline-treated controls. Given the known safety of Tβ4 in clinical trials and its beneficial effects on SCI recovery, the results of this study suggested that Tβ4 is a good candidate for SCI treatment in humans.

Thymosin β10 (Tβ10) expression is associated with malignant phenotypes in many cancers. However, the role and mechanisms of Tβ10 in liver fluke-associated cholangiocarcinoma (CCA) are not fully understood. In this study, we investigated the expression of Tβ10 in CCA tumor tissues and cell lines as well as molecular mechanisms of Tβ10 in tumor metastasis of CCA cell lines.

Thymosin beta family has a significant role in promoting hair regeneration, but which type of T cells play a key role in this process has not been deeply studied. This research aimed to find out the subtypes of T cell that play key role in hair regeneration mediated by thymosin beta 15 (Tβ15).

Although the various biological roles of thymosin β4 (Tβ4) have been studied widely, the effect of Tβ4 and Tβ4-expressing cells in the liver remains unclear. Therefore, we investigated the expression and function of Tβ4 in chronically damaged livers. CCl4 was injected into male mice to induce a model of chronic liver disease. Mice were sacrificed at 6 and 10 weeks after CCl4 treatment, and the livers were collected for biochemical analysis. The activated LX-2, human hepatic stellate cell (HSC) line, were transfected with Tβ4-specific siRNA and activation markers of HSCs were examined. Compared to HepG2, higher expression of Tβ4 in RNA and protein levels was detected in the activated LX-2. In addition, Tβ4 was up-regulated in human liver with advanced liver fibrosis. The expression of Tβ4 increased during mouse HSC activation. Tβ4 was also up-regulated and Tβ4-positive cells were co-localized with α-smooth muscle actin (α-SMA) in the livers of CCl4-treated mice, whereas such cells were rarely detected in the livers of corn-oil treated mice. The suppression of Tβ4 in LX-2 cells by siRNA induced the down-regulation of HSC activation-related genes, tgf-β, α-sma, collagen, and vimentin, and up-regulation of HSC inactivation markers, ppar-γ and gfap. Immunofluorescent staining detected rare co-expressing cells with Tβ4 and α-SMA in Tβ4 siRNA-transfected cells. In addition, cytoplasmic lipid droplets were observed in Tβ4 siRNA-treated cells. These results demonstrate that activated HSCs expressed Tβ4 in chronically damaged livers, and this endogenous expression of Tβ4 influenced HSC activation, indicating that Tβ4 might contribute to liver fibrosis by regulating HSC activation.